Decahydrofluorene alkaloids, processes for their preparation and use in the preparation of antibacterial medicaments

Abstract

The application discloses a decahydrofluorene type alkaloid compound, a preparation method thereof and application thereof in preparation of antibacterial drugs. The structural formula of the alkaloid compound is shown as formula (I). The compound is isolated from mycelium of marine fungi Microascus sp. SCSIO 41821. Experiments prove that 10-13 of the application have the activity of inhibiting protein tyrosine phosphatase PTPSig; compounds 4, 9 and 10 have significant inhibitory activity on 7 strains of bacteria, and the MIC value ranges from 0.1 to 0.8 mu g / mL, which is slightly stronger than that of positive drugs gentamicin sulfate and vancomycin, and the compounds can be applied to protein tyrosine phosphatase inhibitors or antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of marine organisms, and particularly relates to 13 decahydrofluorene type alkaloids and a preparation method and application thereof in preparing antibacterial drugs. BACKGROUND

[0002] Due to wide application, abuse and unreasonable use of antibacterial drugs, emergence and spread of multi-drug resistant bacteria are caused, with increasing drug-resistant pathogenic bacteria, slow development of new antibacterial drugs, long development cycle and slow development speed, so that development of new antibiotics has great significance. It is feasible to find new antibacterial drugs from microbial metabolites, and existing researches show that microbial metabolites are a potential source of new antibacterial agents.

[0003] Decahydrofluorene type alkaloids are a kind of fungal metabolites, which are complex natural alkaloids composed of 1 six, 5, 6 fused tricyclic basic skeleton, 1 gamma-lactam or succinimide heterocycle and 12 or 13 membered ring closely connected with para-aryl ring, and have attracted wide attention due to challenging structure and diverse biological activities (antibacterial, anti-inflammatory, cytotoxic activity). Therefore, the decahydrofluorene type alkaloid compounds have broad application prospects and can be used as potential raw materials of new drugs. SUMMARY

[0004] The first object of the present application is to provide 8 new decahydrofluorene type alkaloids microascones A (1)-B (2), 2, 3-epoxyphomapyrrolidone C (3), 14, 16-epiascomylactam B (4), 24-hydroxyphomapyrrolidone A (5), microascones C-E (6) (7) (8) and known compounds ascomylactams A-B (9) (19), phomapyrrolidones A-C (11) (12) (13) having antibacterial activity or inhibitory effect on protein tyrosine phosphatase.

[0005] The 8 new decahydrofluorene type alkaloids microascones A-B, 2, 3-epoxyphomapyrrolidone C, 14, 16-epiascomylactam B, 24-hydroxyphomapyrrolidone A, microascones C-E and known compounds ascomylactams A-B, phomapyrrolidones A-C of the present application have structural formula as shown in formula (I).

[0006]

[0007] It is a second object of the present application to provide a process for the preparation of compounds microascones A-B, 2,3-epoxyphomapyrrolidone C, 14,16-epiascomylactam B, 24-hydroxyphomapyrrolidone A, microascones C-E, ascomylactams A-B, phomapyrrolidones A-C, which are isolated from the mycelium of the fungus Microascus sp. SCSIO 41821 GDMCC 64206.

[0008] Preferably, the specific steps are as follows:

[0009] (a) preparing the mycelium of the fungus Microascus sp. SCSIO 41821 GDMCC 64206;

[0010] (b) crushing the mycelium obtained in step (a) and soaking with acetone or methanol or dichloromethane:methanol (1:1 v / v), repeatedly extracting, combining the extracts, and concentrating to remove acetone or methanol or dichloromethane and methanol, and extracting the remaining aqueous phase with ethyl acetate, combining the ethyl acetate phases, and concentrating to obtain a mycelium extract;

[0011] (c) subjecting the mycelium extract of step (b) to normal phase silica gel column chromatography, and eluting with a solvent system of dichloromethane:methanol in a volume ratio of 100:0, 98:2, 90:10, 80:20, 70:30, 50:50, 0:100, respectively, in gradient;

[0012] Collecting the sample eluted with dichloromethane:methanol in a volume ratio of 98:2 and 9:1 to obtain component Fr. 2, and subjecting it to normal pressure normal phase silica gel column chromatography, and eluting with a solvent system of petroleum ether:ethyl acetate in a volume ratio of 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 7:3, 6:4, 1:1, 0:1, respectively, in gradient;

[0013] Collecting the sample eluted with petroleum ether:ethyl acetate in a volume ratio of 10:1 to obtain component Fr. 2.3, collecting the sample eluted with petroleum ether:ethyl acetate in a volume ratio of 5:1 to obtain component Fr. 2.4, and collecting the sample eluted with petroleum ether:ethyl acetate in a volume ratio of 4:1 to obtain component Fr. 2.5. Collecting the sample eluted with petroleum ether:ethyl acetate in a volume ratio of 4:1-1:1 to obtain component Fr. 2.6.

[0014] Fractions Fr.2.3 were separated by gel Sephadex LH-20 and purified by HPLC semi-preparative to obtain compounds 6, 7 and 11; fraction Fr.2.5 was separated by medium pressure reverse phase column chromatography eluted with methanol / water in gradient from 13:87 to 100:0, the fraction eluted with methanol / water in 60:40 was collected and separated by medium pressure ODS column to obtain fractions Fr.2.5.1-Fr.2.5.8, Fr.2.5.5 was purified by HPLC semi-preparative to obtain 1; Fr.2.5.7 was purified by HPLC semi-preparative to obtain 4, 8, 10, 12 and 13, compound 4 was recrystallized in methanol to obtain 9;

[0015] Fraction Fr.2.6 was separated by medium pressure ODS column eluted with MeOH / H2O (v / v 50:50-100:0) to obtain 10 fractions, Fr.2.6.2 was purified by HPLC semi-preparative to obtain 2; Fr.2.6.3 was purified by gel and HPLC semi-preparative to obtain 5; Fr.2.6.6 was purified by HPLC semi-preparative to obtain 3.

[0016] Further preferably, the mycelium in step (a) is prepared by the following method: the fungus Microascus sp. SCSIO 41821 is grown in a flat plate culture medium suitable for fungi, after the fungus grows into a colony, the fungus is inoculated into a fermentation medium, and incubated at room temperature for 28 days to obtain the mycelium, the fermentation medium is prepared according to the following proportions per bottle: rice 80 g, yeast extract 0.4 g, glucose 0.4 g, sea salt 3.6 g, 120 ml water.

[0017] Further preferably, the concentration in step (b) is concentrated by reduced pressure.

[0018] A third object of the present application is to provide the use of compound 14, 16-epiascomylactam B, ascomylactams A-B or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug.

[0019] A fourth object of the present application is to provide an antibacterial drug, which contains any one of compounds 14, 16-epiascomylactam B, ascomylactams A-B or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] A fifth object of the present application is to provide a protein tyrosine phosphatase PTP Sig inhibitor, which contains any one of compounds ascomylactam B, phomapyrrolidones A-C or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] The sixth object of the present application is to provide the use of the fungus Microascus sp. SCSIO 41821 GDMCC 64206 in the preparation of compounds microascones A-B, 2,3-epoxyphomapyrrolidone C, 14,16-epiascomylactam B, 24-hydroxyphomapyrrolidone A, microascones C-E, ascomylactams A-B, phomapyrrolidones A-C.

[0022] The compounds 10-13 of the present application have the activity of inhibiting protein tyrosine phosphatase PTPSig, and the compounds 4, 9 and 10 have significant inhibitory activity on 7 strains of bacteria, with the MIC value ranging from 0.1 to 0.8 μg / mL, which is slightly stronger than the positive drug gentamicin sulfate and vancomycin, and these compounds can be used in the application of protein tyrosine phosphatase inhibitors or antibacterial drugs.

[0023] The fungus Microascus sp. SCSIO 41821 of the present application was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on December 27, 2023, and the address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, and the preservation number is GDMCC No: 64206. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Key COSY, HMBC correlations for compounds 1-8 (compounds microascones A-B, 2,3-epoxyphomapyrrolidone C, 14,16-epiascomylactam B, 24-hydroxyphomapyrrolidone A, microascones C-E);

[0025] Figure 2 Key NOESY correlations for compounds 1-8 (compounds microascones A-B, 2,3-epoxyphomapyrrolidone C, 14,16-epiascomylactam B, 24-hydroxyphomapyrrolidone A, microascones C-E).

[0026] Figure 3 Single crystal structure for compound 9 (ascomylactam A) DETAILED DESCRIPTION

[0027] The following examples are further illustrations of the application and are not intended to limit the same.

[0028] Example 1

[0029] (1) The fermentation medium was prepared by mixing rice 80 g, yeast extract 0.4 g, glucose 0.4 g, sea salt 3.6 g, and 120 ml water in the ratio of 80:0.4:0.4:3.6:120. In this way, 66 bottles of fermentation medium were prepared and sterilized for later use.

[0030] (2) Preparation of mycelium: The fungus Microascus sp. SCSIO 41821 was grown in a potato water agar plate medium suitable for fungi. After the fungus grew into a colony, the fungus was inoculated into a rice fermentation medium (composition: 80 g rice, 0.4 g glucose, 0.4 g yeast extract, 3% sea salt, 120 ml water, sterilized) and incubated at room temperature for 28 days to obtain mycelium. The mycelium was crushed and soaked in acetone for 3 times overnight, and then extracted with CH2Cl2 / MeOH (volume ratio 1:1) mixed solvent for 3 times by ultrasonic extraction. The filtrate was obtained by filtering the mycelium, and the water part was concentrated under reduced pressure to remove the solvent, and then extracted with ethyl acetate for 5 times. The concentrated mycelium extract was obtained in an amount of 117.8 g.

[0031] (3) Compound separation and purification: The mycelium extract was dry-mixed with normal phase silica gel (100-200 mesh), and then loaded into a glass chromatography column (H fine silica gel). Normal pressure column chromatography was performed at room temperature, and the column was eluted with solvent systems with gradient elution of dichloromethane:methanol in the volume ratio of 100:0, 98:2, 90:10, 80:20, 70:30, 50:50, and 0:100, respectively. The results of TLC and HPLC analysis were combined, and 7 fractions Fr.1-Fr.7 were obtained.

[0032] The sample eluted with dichloromethane:methanol in the volume ratio of 98:2 and 9:1 was collected to obtain fraction Fr.2. Fr.2 (40.5 g) was subjected to normal pressure column chromatography on normal phase silica gel, and the column was eluted with solvent systems with gradient elution of petroleum ether:ethyl acetate in the volume ratio of 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 7:3, 6:4, 1:1, and 0:1, respectively. The sample eluted with petroleum ether:ethyl acetate in the volume ratio of 10:1 was collected to obtain fraction Fr.2.3, the sample eluted with petroleum ether:ethyl acetate in the volume ratio of 5:1 was collected to obtain fraction Fr.2.4, and the sample eluted with petroleum ether:ethyl acetate in the volume ratio of 4:1 was collected to obtain fraction Fr.2.5. The sample eluted with petroleum ether:ethyl acetate in the volume ratio of 4:1-1:1 was collected to obtain fraction Fr.2.6.

[0033] Component Fr.2.3.4 obtained from the gradient elution of the column with solvent system of petroleum ether: ethyl acetate in the ratio of 20:1 was separated on Sephadex gel LH-20 with eluent CH2Cl2: CH3OH 1:1 to obtain Fr.2.3.4-2 which was purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm; eluent CH3CN / H2O / TFA (v / v / v 94:6:0.03) at a flow rate of 3 ml / min) to obtain compound 11 (37.0 mg, t R = 25.5 min); Fr.2.4 (8.32 g) was separated on Sephadex gel LH-20 with eluent CH2Cl2: CH3OH 1:1 to obtain Fr.2.4.3 which was purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm; eluent CH3CN / H2O / TFA (v / v / v 94:6:0.03) at a flow rate of 3 ml / min) to obtain compound 6 (1.2 mg, t R = 27.8 min), 7 (1.4 mg, t R = 31.0 min); Component Fr.2.5 (1.38 g) was subjected to medium pressure reverse phase column chromatography eluting with acetonitrile / water in the ratio of 50:50 to 100:0 to obtain Fr.2.5.1 to Fr.2.5.8. The sample eluting with acetonitrile: water in the ratio of 67:33 gave component Fr.2.5.5 which was purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm; eluent CH3CN / H2O (v / v 81:19, 3 mL / min) to obtain compound 1 (2.7 mg, t R = 28.0 min); The sample eluting with acetonitrile: water in the ratio of 72:28 gave component Fr.2.5.7 which was purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm; eluent CH3CN / H2O (v / v 81:19, 3 mL / min) to obtain compound 4 (10.0 mg, t R = 64.5 min), compound 8 (2.3 mg, t R = 30.6 min), 10 (5.0 mg, t R = 21.3 min), compound 12 (3.5 mg, tR = 35.0 min) and compound 13 (5.5 mg, t R = 26.1 min), compound 4 was recrystallized in methanol to give compound 9;

[0034] Fraction Fr.2.6 (8.32 g) was subjected to medium pressure ODS system with MeOH / H2O (v / v 50:50-100:0) to give 10 fractions. Fraction Fr.2.6.2 was collected from the sample eluted with methanol:water volume ratio of 72:28 and purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm) with CH3OH / H2O / TFA (v / v / v 76:24:0.01) at a flow rate of 3 ml / min to give compound 2 (3.7 mg, t R = 24.2 min); Fraction Fr.2.6.3 was collected from the sample eluted with methanol:water volume ratio of 72:28 and purified by gel washed with methanol and semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm) with CH3OH / H2O (v / v 80:20) at a flow rate of 3 ml / min to give compound 5 (1.5 mg, t R = 25.7 min); Fraction Fr.2.6.6 was collected from the sample eluted with methanol:water volume ratio of 81:19 and purified by semi-preparative HPLC (YMC-Pack ODS column, 250 x 10 mm, S-5 μm, 12 nm) with CH3CN / H2O (v / v 67:33, 3 mL / min) to give compound 3 (4.3 mg, t R = 34.0 min).

[0035] Structure assignment:

[0036] NMR data of compounds 1-13 are shown in Tables 1-2.

[0037] Compound 1 is a white powder, and shows a quasi-molecular ion peak m / z 528.3109 [M+H] + in high resolution mass spectrum, combined with NMR data, the molecular formula of compound 1 is deduced as C 34 H 41 N04, with 15 degrees of unsaturation. There are 4 aromatic hydrogen signals δ H 7.01 (dd, J = 8.8, 2.0 Hz, H-25), 6.94 (dd, J = 8.7, 2.6 Hz, H-26), 6.86 (dd, J = 8.1, 2.0 Hz, H-29), 6.72 (dd, J = 8.1, 2.6 Hz, H-28), and 5 methyl signals δ H1.68 (s, H3-30), 1.65 (s, H3-32), 1.32 (s, H3-31), 1.06 (d, J = 6.2 Hz, H3-33), 0.90 (d, J = 6.6 Hz, H3-34); 13 The C NMR and DEPT spectra showed the structure contained 34 carbons, including 2 carbonyl carbons δ C 188.1, 174.7, 7 quaternary carbons, 4 methylenes, 16 methines, 5 methyl carbons. By comparing the carbon and proton NMR data, compound 1 was found to be similar to embellicines

[61] , phomapyrrolidones

[63] and ascomylactams

[64] It was speculated that this compound was a decahydrofluorene type alkaloid.

[0038] Detailed analysis of the 2D spectra (HSQC, HMBC, COSY) of compound 1 suggested that the structure contained the same A / B / C and G / H rings as ascomylactam B (10). H3-22 (δ H 1.32) was correlated to C-4 / C-5 / C-6 / C-17 in the HMBC spectrum, H-17 (δ H 3.21) was correlated to C-5 / C-15 / C-16 / C-18, H3-21 (δ H 1.68) was correlated to C-2 / C-3 / C-4, H-4 (δ H 2.49) was correlated to C-2 / C-3 / C-19, H-2 (δ H 5.38) was correlated to C-1 / C-4 / C-1’, H-1 (δ H 2.07, 1.86) was correlated to C-2 / C-3 / C-19 / C-1’, H-1’ (δ H 2.53) was correlated to C-1 / C-18 / C-19 / C-20 / C-2’, and NH (δ H 8.38) was correlated to C-19 / C-20 / C-1’ / C-2’, in combination 1 H- 1 H COSY spectrum, H-1 was correlated to H-2 / H-1’ ( Figure 1 ), which determined the D / E / F ring system, thus the planar structure of compound 1. The unusual D / E / F tri-ring system in the structure was believed to be formed by a Diels-Alder reaction between the double bond of the γ-lactam moiety and the 1,4-pentadienyl group at C-5. Unlike other alkaloids, the ring D / E / F in 1 forms a novel tri-fused ring skeleton, thus creating a 6 / 5 / 6 / 5 / 6 / 5 / 13 / 6 polycyclic system.

[0039] In the NOESY spectrum, H-4 was correlated with H-17 / H3-22, H-17 with H-8 / H-15 / H-6', H-15 with H-8 / H-14 / H-6' / H3-25, H-14 with H-10α / H-12α, NH with H-3'α / H-5', H-3'α with H-5', OH-6' with H-1' NOE, indicating that these hydrogens are on the same side of the plane. In addition, H-12β was correlated with H-9 / H-10β, H-10β with H3-23, H-13β with H-16, H-16 with H-8', H-9' with H-3'β NOE, indicating that the above hydrogen atoms are on the other side of the plane, which preliminarily determines the relative configuration of compound 1. The absolute configuration of compound 1 is finally determined by ECD chemical calculation. Since the relative configuration of C-19 is not determined, three model compounds (19R,1'S)-1a, (19S,1'R)-1b, (19S,1'S)-1c of 1 are calculated, and the model compound (19R,20R)-1d cannot be calculated ECD because of unstable configuration. By comparing the ECD diagrams of the three models, it is found that (19S,1'S)-1c is consistent with the measured ECD diagram, so the absolute configuration of compound 1 is determined as 4S,5R,8S,9S,11R,13S,14R,15R,16S,17R,19S,1'S,2' R, named as microascone A.

[0040] Compound 2 is a white powder, and the high-resolution mass spectrum gives a quasi-molecular ion peak m / z 544.3058 [M+H] + , and the NMR data in Table 1-2 is speculated to be C 34 H 41 NO5, with 15 degrees of unsaturation. The NMR data of compound 2 is very similar to that of 1, the difference is that there is a methylene signal (δ H 3.24) and a active hydrogen signal (δ H 4.40) in the hydrogen spectrum of 2, and one methyl signal is less; 13 C NMR and DEPT spectrum shows that there is one more oxidized methylene in the structure δ C66.4, missing one methyl carbon. By detailed analysis of the 2D spectra (HSQC, HMBC, COSY) of compound 2, it was found that it has the same A-G ring as 1, the difference between them is that the methyl group at C-24 of 2 is oxidized. H-24 is related to H-11, OH-24 in COSY spectrum, and HMBC spectrum shows that H2-24 is related to C-9 / C-10 / C-11, confirming that the methyl group at C-24 is oxidized to methylene. Combined with other signals of 2D spectra, the planar structure of compound 2 was determined. The NOESY spectrum signals of compound 2 are basically the same as those of compound 1, and it is speculated that the relative configuration of compound 2 is the same as that of compound 1. By comparing the experimental ECD spectra of compound 2 and 1, and the theoretical calculation ECD spectra of model compound (4S, 5R, 8S, 9S, 11R, 13S, 14R, 15R, 16S, 17R, 19S, 1’S, 2’R)-1c, it was found that they are basically consistent in trend, thereby determining the absolute configuration of compound 2.

[0041] Compound 3: white powder, the quasi-molecular ion peak m / z 560.3002 [M+H] was observed in high resolution mass spectrum + , combined with NMR data (Tables 1-2), it is speculated that the molecular formula is C 34 H 41 NO6. These NMR data are very similar to phomapyrrolidone C (13), the difference is that 3 has one more methine in the hydrogen spectrum (δ H 2.99, 1H) and two less olefinic hydrogen protons; in the carbon spectrum, it has one more oxygen-containing methine (δ C 69.6) and one more quaternary carbon (δ C 66.9), and two less olefinic carbons, it is speculated that the double bond of the D ring disappears in the structure. In the HMBC spectrum ( Figure 1 ), H-3 (δ H 2.99) is related to C-2 / C-4 (δ C 47.1) / C-5 (δ C 141.9), H3-20 (δ H 1.00) is related to C-1 / C-2 / C-5, H3-21 (δ H 1.34) is related to C-1 / C-2 / C-3, and H3-22 (δ H 0.64) is related to C-3 / C-4 / C-5 / C-16, indicating that there is an epoxy bond between C-2 and C-3, instead of a double bond. Detailed analysis of the 2D spectra of the compound determines the planar structure of the compound.

[0042] NOESY spectra showed that H-16 was correlated with H-1 / H-1' / H-8' / H-9', H-15 was correlated with H-9β / H-1' / H-8' / H-9', H-1 was correlated with H3-21, and H-3 was correlated with H3-21, indicating that these hydrogens were on the same side of the plane; according to the correlation of H-14 with H-7 / H-6' / H3-25, H-7 with H3-22, and H-9α with H-13 / H3-25, it was determined that they were on the other side of the plane, and the relative configuration of the compound was preliminarily determined. By comparing the measured ECD of compound 3 with that of phomapyrrolidone C, it was found that the CD curve trends of the two were very similar. In addition, the theoretical ECD of (1R, 2R, 3S, 4R, 7S, 8S, 10R, 12S, 13R, 14R, 15S, 16S, 18R, 1'R, 2'R)-3 was calculated by Gaussian software, and it was found that the measured ECD was consistent with the calculated ECD graph, thereby determining the absolute configuration of the compound. The identified structure is shown in the figure, and is named 2,3-epoxyphomapyrrolidone C.

[0043] Compound 4 was a white powder, and the quasi-molecular ion peak m / z 528.3130 [M+H] was shown in the high-resolution mass spectrum + , and the molecular formula was inferred to be C 34 H 41 NO4 with 15 degrees of unsaturation based on the NMR data (Tables 1-2). The NMR data of compound 4 were very similar to those of ascomylactam B, embellicine A and other compounds, and it was inferred that 4 also belonged to this type of alkaloid compound. Detailed analysis of the two-dimensional spectra (HSQC, HMBC and COSY) of compound 4 basically determined the planar structure of compound 4, which was the same as that of ascomylactam B and embellicine A, but since the chemical shifts of C-13, C-14, C-15 and C-16 of compound 4 deviated greatly from those of ascomylactam B and embellicine A, it was inferred that this might be due to the different configurations of C-14 or C-16, and it was preliminarily inferred that they were epimers.

[0044] The relative configuration of compound 4 was determined by NOESY spectrum, according to the NOE correlation signals of H-7 with H-9a / H-13 / H-14 / H3-22, H3-22 with H3-20, H-9a with H-13, H-14 with H-6', which indicated that these hydrogens were on the same side of the plane; the NOE correlation signals of H-1 with H3-23, H-8 with H-15, H-12 with H-9b / H-10, H-15 with H-8', H-16 with H-8', H-19 with H-3 / H-16 in NOESY spectrum, which indicated that they were on the other side of the plane. According to the reported NOE correlation signals, the relative configuration of chiral centers at C-14 and C-16 in the structure was found to be the same as ascomylactam A (9) and embellicine A, and opposite to ascomylactam B (10). From the above, it can be seen that the configuration of C-14 and C-16 of compound 4 is different from ascomylactam B. Then the ECD spectrum of model compound (1S,4R,7S,8S,10R,12S,13R,14R,15S,16S,2'R)-4 was calculated by Gaussian software, and it was found that the theoretically calculated ECD spectrum was in good agreement with the measured CD spectrum, thereby determining the absolute configuration of compound 4. In fact, during the separation and purification process, it was found that compound 4 was unstable due to the easy change of enolic and ketonic forms of C-17 and C-18. In addition, 4 can obtain colorless crystal ascomylactam A (9) in methanol, which further confirms that the absolute configuration of 4 is 1S,4R,7S,8S,10R,12S,13R,14R,15S,16S,2'R, named as 14,16-epiascomylactam B.

[0045] Compound 5 is a white powder, and the high resolution mass spectrum gives a quasi-molecular ion peak m / z 544.3060 [M+H] + , and the molecular formula of 5 is deduced as C 34 H 41 NO5 according to the NMR data in Table 1-2. The NMR data of 5 is very similar to phomapyrrolidone A (11), except that 5 has one less methyl signal and one more oxymethylene signal (δ C 66.9; δ H 3.25) and one alcohol hydroxyl (OH-24, δ H 4.40) signal in the hydrogen spectrum and carbon spectrum of 5. In the COSY spectrum, H2-24 (δ H3.25) Correlation with H-11 and OH-24: In the HMBC spectrum, H2-24 is correlated with C-9 / C-10 / C-11, confirming that the hydroxyl group is connected to C-24, indicating that the methyl group at C-33 in structure 11 is oxidized to a methylene group. Detailed analysis of HMBC and COSY spectra confirmed the planar structure of compound 5.

[0046] In the NOESY spectrum ( Figure 2 The results show that H-7 is correlated with H-9α / H-13 / H-14 / H3-22, H-13 is correlated with H-7, H-14 is correlated with H-7 / H3-25, H-16 is correlated with H-13 / H-18 / H-6', and H3-20 is correlated with H3-25, indicating that these groups are on the same side of the plane. In addition, H-12 in the NOESY spectrum is correlated with H-8 / H-15, H-15 is correlated with H-1' / H-8', and H-1' is correlated with H-3 / H-9', these NOE correlation signals indicate that they are on the other side of the plane. Further calculations of the ECD of compound 5 were performed to determine its absolute configuration. The theoretically calculated ECD of (1S,4R,7S,8S,10R,12S,13R,14R,15S,16R,18R,1'S)-5 was consistent with the experimental CD curve, thus determining the absolute configuration of 5 as shown in formula (I), and naming it 24-hydroxyphomapyrrolidone A.

[0047] Compound 6 is a white powder, and high-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 528.3103 [M+H]. + Based on the NMR data, its molecular formula can be deduced to be C. 34 H 41 NO4 has 15 degrees of unsaturation. 1 H and 13 The C10 NMR spectra are very similar to those of phomapyrrolidone A (11). The main difference is that compound 6 has an additional substituted double bond (δ¹⁰) in its proton and carbon spectra. H 5.03, 1H, d, J = 10.9 Hz, δ H 4.98, 1H, dd, J = 17.5, 1.1 Hz, δ H 6.45,1H,dd,J=17.5,10.9Hz; δ C 114.1,CH2,δ C The structure (135.6, CH) lacks one methine and one methyl signal in the high field, suggesting that the D ring of phomapyrrolidone A undergoes ring opening at C-1 and C-5 positions. Detailed analysis of the compound's two-dimensional spectrum confirmed this hypothesis. The COSY spectrum shows a correlation between H-1 and H-2, while the HMBC spectrum (…) Figure 1) Display H-1(δ H 5.03, 1H, d, J = 10.9 Hz, δ H 4.98, 1H,dd,J=17.5,1.1Hz) are related to C-2 / C-3, H-2 (δ H 6.45, 1H, dd, J=17.5, 10.9Hz) is associated with C-3 / C-4, H3-21 is associated with C-2 / C-3 / C-4, H3-21 is associated with C-4 / C-5 / C-6 / C-17, and H-4 is associated with C-2 / C-3 / C-5 / C-6 / C-17 / C-22. This confirms the presence of a 3-methylbutadiene fragment in the structure of compound 6, which is attached to the C-5 position. Thus, the planar structure of compound 6 is determined as shown in formula (I).

[0048] In the NOESY spectrum ( Figure 2 The correlations between H-8 and H-10α / H-14 / H-17, H-12α and H-14 / H3-25, H-15 and H-17 / H-19 / H-6', H-17 and H-8 / H3-22, and H-19 and H-5' indicate that these hydrogens are located on the same side of the plane. The correlations between H-11 and H-13, H-16 and H-2 / H-9 / H-8', and H-20 and H-9' suggest that these hydrogens are located on the other side of the plane. Furthermore, the NOE correlation between H-2 and H3-32 confirms that the double bond configuration at the C-3 and C-4 positions is Z-configuration. The absolute configuration of compound 6 was further determined by ECD calculation. The theoretical ECD of (5R,8S,9S,11R,13S,14R,15R,16S,17R,19R,1'S)-6 matched well with the measured ECD spectrum, thus determining the absolute configuration of compound 6 and identifying the structure as microascone C.

[0049] Compound 7 is a white powder, and high-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 528.3115 [M+H]. + Based on the NMR data, its molecular formula can be deduced to be C. 34 H 41 NO4 has 15 degrees of unsaturation. 1 H and 13 The C10 NMR spectrum is very similar to that of compound 6, the difference being the presence of H-1 (δ) in the proton NMR spectrum of compound 7. H 4.94, 4.76), H-2(δ H 5.91), H-4(δ) H 4.96) and H-30 (δ) H 1.42) Chemical shift and δ of 6 H-1 5.03, 4.98; δ H-2 6.45; δ H-44.87; δ H-4 The difference is obvious at 1.45), with C-1 (δ) in the carbon spectrum showing significant differences. C 111.2), C-2(δ C 141.0), C-3(δ C 135.1), C-4(δ C 142.5) and C-30 (δ) C The chemical shift of 13.6) and the (δ) of 6 C-1 114.1; δ C-2

[0050] 135.7; δ C-3 131.6; δ C-4 138.0; δ C-30 20.9) Significant differences were observed. Detailed analysis of the two-dimensional spectra (HSQC, COSY, and HMBC spectra) confirmed the planar structure of compound 7, which is identical to that of compound 6. However, comparison of the C-H spectra of these two compounds revealed shifts in the chemical shifts at the C-1 / C-2 / C-3 / C-4 positions. It is speculated that the difference in chemical shifts may be due to configurational changes in the double bonds at the C-3 and C-4 positions in compound 7, rather than a configurational change in the chiral center at the C-5 position. In the NOESY spectrum, H3-21 is correlated with H-1 / H-6, indicating that the double bond configuration between C-3 and C-4 is E. Based on the NOESY spectrum ( Figure 2 The relevant signals revealed that the relative configuration of 7 is the same as that of 6. Furthermore, the experimental ECD spectra of compounds 6 and 7 are very similar, indicating that the absolute configuration of 7 is the same as that of 6.

[0051] Compound 8 is a white powder, and high-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 548.3363 [M+H]. + Based on the NMR data, its molecular formula can be deduced to be C. 34 H 46 NO5. Compound 8 1 H and 13CNMR data was similar to compound 4, the main difference was that there was one less trisubstituted double bond signal and one less quaternary carbon in the carbon spectrum, and one more carbonyl carbon, one more methylene and one more methoxy. In the HMBC spectrum, H-16 was associated with C-17 / C-18, H-18 was associated with C-17 / C-19, OCH3-19 was associated with C-19, H-3' was associated with C-2' / C-4' / C-5', indicating that the lactam ring and the 13-membered macrocycle of compound 4 were opened in 8. Detailed analysis of the HMBC and COSY correlation signals of the compound determined the planar structure of compound 8. Its relative configuration was determined by analyzing the NOESY spectrum, and then the absolute configuration of 8 was determined by ECD calculation. The theoretical ECD curve of (1S,4R,7S,8S,10R,12S,13R,14R,15S,16R)-8 was in good agreement with the experimental CD curve trend, thereby identifying the structure as shown in the figure.

[0052] Compound 10 is a white powder, 1 Four para-coupled aromatic protons appeared in the H NMR spectrum δ H 7.04 (d, J = 8.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 1.4 Hz, 1H), 6.72 (dd, J = 8.2, 2.4 Hz, 1H), 6 methyls δ H 1.73 (s, 3H), 1.45 (s, 3H), 1.34 (s, 3H), 0.94 (d, J = 7.0 Hz, 1H), 0.92 (d, J = 7.0 Hz, 3H), 2 active hydrogen signals δ H 6.39 (s, 1H), 8.38 (s, 1H); 13 The C NMR spectrum showed that the compound had 34 carbons, including 2 carbonyl carbons δ C 196.5, 168.5, 6 methyls 29.1, 18.6, 15.1, 14.5, 22.4, 20.2. 1 H and 13 The C NMR data was similar to compound 4, the main difference was that the chemical shifts of C-14 and C-15 were different. By comparing the spectral data, it was found that the data of compound 10 was basically consistent with the literature ascomylactam B, and the structure was identified as shown in formula (I).

[0053] Compound 11: white powder, 1 H and 13 The C NMR data showed that the compound was very similar to 10, the hydrogen spectrum of 10 had one less olefinic hydrogen and two more methine (δ H 2.93 (d, J = 2.1 Hz, 1H); 3.44, m), and the carbon spectrum suggested that there were two more high-field methine carbons in the structure δ C59.7, 43.4 and 1 carbonyl carbon δ C 178.3, less 1 olefinic secondary carbon and 2 quaternary carbons. By comparing with the literature, it was found that the NMR data of this compound was basically the same as that of phomapyrrolidone A. In 2019, Chen et al. corrected the configuration of phomapyrrolidone A by single crystal method, and the corrected structure is shown as formula (I).

[0054] Compound 12: white powder, high resolution mass spectrometry (HRESI-MS) gave ion peak m / z 528.3110 [M+H] + , combined with NMR data (Table 1-2), the molecular formula was speculated to be C 35 H 43 NO4, there were 15 unsaturations. The NMR data of compound 12 was very similar to that of 11, mainly the carbon and hydrogen chemical shifts of C-13 / C-14 / C-15 / C-16 / C-18 / C-1' positions were changed, which was speculated to be different chiral carbon configurations, and they might be epimers. By comparing with the literature, it was found that the spectral data of 12 was basically the same as that of phomapyrrolidone B reported, and the compound was identified as phomapyrrolidone B.

[0055] Compound 13: white powder, 1 H and 13 The NMR data of compound 13 was very similar to that of compound 11. In the NMR data of compound 13, one more active hydrogen signal and one oxygen-containing methine were found than in 11, and one carbonyl signal was less, which was speculated to form an epoxide bond between C-18 and C-1'. By further comparing the NMR data, it was found that 13 was basically the same as phomapyrrolidone C in the literature. In 2019, the configuration of phomapyrrolidone C was corrected, and the corrected structure is shown as formula (I).

[0056] Table 1. 1 The NMR data of compounds 1-8 1 HNMR data (DMSO-d6, δ H in ppm, J in Hz)

[0057]

[0058]

[0059]

[0060]

[0061] a500 MHz for 1 H NMR. b 700 MHz for 1 H NMR.

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] a 125MHz for 13 C NMR. b 175MHz for 13 CNMR. c DetectedbyHMBC correlations.

[0068] Antibacterial activity test of compounds 1-13 in Example 2

[0069] The MIC value of the compounds with significant antibacterial activity in the preliminary screening was determined by the half dilution method. (1) Preparation of sample solution: 100 μL of LB culture solution was added to each well of a 96-well plate, 2 μL of 20 mg / mL sample was added to the first well, 98 μL of LB culture solution was added, mixed, 100 μL of sample solution was taken into the second well, mixed, and the operation was repeated in sequence, so that the sample solution in each well was diluted to different concentrations; (2) the bacterial solution of the indicator bacteria was diluted with LB culture medium to OD 600 = 0.01-0.05 to prepare a bacterial suspension; (3) 100 μL of diluted bacterial solution was added to each well, and the sample was finally prepared to 100, 50, 20, 10, 5.0, 2.0, 1.0 μg / mL, negative controls (LB culture medium + DMSO + bacterial solution), positive controls (positive drug concentration of 0.5-50 μg / mL), blank controls (LB culture medium) were set for each well plate, and incubated at 37°C for 12 h; (4) the OD 600 values at 0 h, 12 h and 24 h were tested by an enzyme marker, and the bacterial growth in the well plate was observed by naked eye, and the experimental results were recorded.

[0070] The 96-well plate half-dilution method was used to further test the MIC values of the compounds 4, 9, 10 with better activity, and the experimental method and operation were the same as above. The sample solution was finally prepared into 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 μg / mL, and the positive drug concentration was 0.039-2.5 μg / mL. It was placed in a 37°C incubator for 12 h and 24 h, and finally the OD 600 values at 0 h, 12 h and 24 h were tested by an enzyme marker, and the bacterial growth in the well plate was observed by naked eye, and the experimental results were recorded.

[0071] The antibacterial activity results of compounds 1-13 (Table 3) showed that the inhibitory activity of compounds 4, 9, 10 on 7 strains of bacteria was slightly stronger than that of the positive drug gentamicin sulfate and vancomycin, and the MIC value range was 0.1-0.8 μg / mL, 3 significantly inhibited the activity of Staphylococcus aureus, and the MIC value was 12.5 μg / mL.

[0072] Antifungal activity test of compounds 4, 9, 10-13 against Candida albicans

[0073] The microdilution method was used. Candida albicans was first activated and cultured in Sabouraud solid medium, and single colonies were picked into Sabouraud liquid medium, and cultured at 30°C, 200 rpm / min on a shaking table for 12 h. The bacterial solution was diluted to OD 600 = 0.01-0.05 with Sabouraud medium to prepare a suspension; the experimental method and operation were the same as above, and the sample solution to be tested was finally prepared into 100, 50, 25, 12.5, 6.2, 3.1, 1.6 μg / mL, and the positive drug concentration was 0.3-20 μg / mL. It was placed in a 37°C incubator for 12 h and 24 h, and finally the OD 600 values at 0 h, 12 h were tested by an enzyme marker, and the bacterial growth in the well plate was observed by naked eye, and the experimental results were recorded.

[0074] Table 3. Antifungal activity of compounds 1-13 and compounds 4, 9-13

[0075]

[0076] “-”: Not tested

[0077] Example 3 Inhibition of protein tyrosine phosphatase activity test of compounds 2-3, 5-7, and 10-13

[0078] Experimental model: five models of protein tyrosine phosphatase (PTP1B, CDC25B, LAR, VHR and PTP Sig)

[0079] Experimental method: (1) The amount of PTP1B and CDC25B enzyme in the reaction system was determined by using enzyme standard curve. The amount of each enzyme in the screening system was: PTP1B (1 μL) and CDC25B (1 μL), and the enzyme screening system was established for screening. (2) The reliability of the model was verified by using positive drug sodium orthovanadate, and the results showed that sodium orthovanadate had obvious inhibitory effect on PTP1B, CDC25B and PTPSig model, and the IC 50 value of sodium orthovanadate was 20.1 μM. (3) Protein tyrosine phosphatase activity test: the test compound was diluted, the initial concentration was set to 5 mM, 2-fold dilution was performed for 8 concentrations, the second concentration was used for primary screening, and the active compound was gradiently re-screened; in a 96-well plate, 2 μL of different concentrations of test sample solution was added, 98 μL of reaction buffer and different protein tyrosine phosphatase liquid (PTP1B enzyme 1 μL + pNPP 0.5 μL; CDC25B enzyme 0.1 μL + pNPP 0.5 μL; PTPSig enzyme 0.3 μL + pNPP 0.5 μL) was added to each well, and Na3VO4 was used as a positive control. After pre-incubation at room temperature for 15 minutes, 1.0 μL of buffer containing 50 mM p-nitrophenyl phosphate (pNPP) substrate was added, and the reaction system was prepared, then incubated at 37°C for 30 min, the luminescence value at 405 nm was detected, the data was counted, and the IC 50 value was calculated by using Graphpad.6, and each sample was repeated three times.

[0080] The test results (Table 4) showed that compounds 10-13 could significantly inhibit PTPSig, the IC 50 value was in the range of 4.5-8.4 μM, compound 10 had certain inhibitory activity on VHR, the IC 50 value was 38.3 μM; compounds 12-13 had obvious inhibitory activity on PTP1B enzyme, the IC 50 values were 10.8 and 8.7 μM, respectively, and 13 could significantly inhibit the activity of CDC25B, the IC 50 value was 6.0 μM.

[0081] Table 4. Inhibitory activity of compounds 2-3, 5, 10-13 on PTPs

[0082]

[0083]

[0084] “-”:Not tested

[0085] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. Any compound as shown in formula (Ⅰ): Equation (Ⅰ).

2. A method for preparing the compound according to claim 1, characterized in that, From fungi Microascus It was isolated from the mycelium of sp.SCSIO 41821 GDMCC 64206; the steps included are as follows: (a) Preparation of fungi Microascus Mycelium of sp. SCSIO 41821 GDMCC 64206; (b) After the mycelium obtained in step (a) is broken, it is soaked in acetone or methanol or dichloromethane:methanol 1:1 v / v, and the extraction is repeated. The extracts are combined and concentrated to remove acetone or methanol or dichloromethane and methanol. The remaining aqueous phase is extracted with ethyl acetate. The ethyl acetate phases are combined and concentrated to obtain the mycelium extract. (c) The bacterial extract described in step (b) was subjected to normal-phase silica gel column chromatography and eluted sequentially with a solvent system of dichloromethane:methanol volume ratios of 100:0, 98:2, 90:10, 80:20, 70:30, 50:50, and 0:

100. Samples washed with dichloromethane:methanol at volume ratios of 98:2 and 9:1 were collected to obtain fraction Fr.2, which was then subjected to normal-phase silica gel column chromatography at ambient pressure, using a gradient elution system with petroleum ether:ethyl acetate volume ratios of 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 4:1, 7:3, 6:4, 1:1, and 0:

1. Component Fr.2.3 was obtained by rinsing samples with a petroleum ether:ethyl acetate volume ratio of 10:1; component Fr.2.4 was obtained by rinsing samples with a petroleum ether:ethyl acetate volume ratio of 5:1; component Fr.2.5 was obtained by rinsing samples with a petroleum ether:ethyl acetate volume ratio of 4:1 to 1:1; and component Fr.2.6 was obtained by rinsing samples with a petroleum ether:ethyl acetate volume ratio of 4:1 to 1:

1. Fraction Fr.2.5 was subjected to medium-pressure reversed-phase column chromatography with a methanol / water gradient elution of 13:87-100:0 (v / v). The sample eluted with methanol:water at a v / v ratio of 60:40 was collected and then separated by medium-pressure ODS column chromatography to obtain fractions Fr.2.5.1-Fr.2.5.

8. Fraction Fr.2.5.7 was purified by semi-preparative HPLC to obtain fractions 4 and 12. The mycelium described in step (a) is prepared by the following method: fungi Microascus sp. SCSIO 41821 was grown on a suitable agar plate medium for fungi. After the fungi emerged, they were inoculated into a fermentation medium and cultured statically at room temperature for 28 days to obtain mycelium. Each bottle of the fermentation medium was prepared according to the following proportions: 80 g rice, 0.4 g yeast extract, 0.4 g glucose, 3.6 g sea salt, and 120 ml water.

3. The preparation method according to claim 2, characterized in that, The concentration described in step (b) is performed using vacuum concentration.

4. The use of compound 12 of claim 1 or a pharmaceutical salt thereof in the preparation of a protein tyrosine phosphatase PTPSig inhibitor.

5. The use of compound 4 of claim 1 or its pharmaceutical salt in the preparation of an antibacterial drug, wherein the antibacterial drug is a drug that inhibits Staphylococcus aureus, methoxystaphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptococcus dolphinus, Streptococcus agalactiae, Bacillus amyloliquefaciens, and Candida albicans.

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