WO2015124037A1 - Compound with four-ring fused structure, preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to the technical field of medicine. The present invention provides a compound with a four-ring fused structure, the chemical structure of which is represented by formula (I). The present invention also provides a preparation method for the compound and an application of the compound in the preparation of antitumor drugs, antifungal drugs, antibacterial drugs, anti-inflammatory drugs and PTP1B inhibitors.

Description

Compound with four-ring ring structure and preparation method and application thereof Technical field

The invention relates to the technical field of medicine, in particular to a novel skeleton compound having a 6/6/5/7 tetracyclic ring structure, a preparation method thereof and application thereof in antitumor, antibacterial and PTP1B inhibitory activities.

Background technique

Compounds with cyclopentane and polyhydrophenanthrene as the basic nucleus are widely found in nature, and their structural formula is tetracyclic ring (A/B/C/D, carbon number 6/6/6/5 respectively). ), mainly includes two major compounds, steroids and tetracyclic triterpenes. In vivo, these two compounds are formed by squalene cyclization through different routes. In addition to the tetracyclic nucleus, the structural formula of the steroid compound often contains 3β-OH, two horn methyl groups (C-10 and C-13 positions) and a hydrocarbon side chain (C-17 position, including C8~). The hydrocarbyl group of C10), which is an important component of the cell membrane of an organism, is largely distributed in animals, plants and microorganisms. The tetracyclic triterpene compound is composed of 6 isoprene units, and its structural formula is based on the tetracyclic nucleus, usually containing 3β-OH, 5 角 methyl groups (2x C-4 positions, C-10 positions, C-13 and C-14) and one hydrocarbyl side chain (C-17 position, C8-C10-containing hydrocarbon group) are mainly present in higher plants and sea cucumbers, and are mostly present in the form of saponins. This family of compounds with a cyclopentane and polyhydrophenanthrene core structure has very rich biological activities such as hormonal action, anti-inflammatory, anti-tumor, anti-fertility and enhanced immunity, and many of them have become widely used drugs in clinical practice (Editor Wu Lijun) Natural Medicine Chemistry, 4th edition, People's Health Publishing House, 2003, 271-349; Zhang Wen, editor, Introduction to Marine Medicine, 2nd Edition, Shanghai Science and Technology Press, 2012, 110-148).

A few compounds of this family have been derived from the enzymatic modification in vivo or the structural modification of chemists on the basis of the mother-nuclear structure of cyclopentane and polyhydrophenanthrene, resulting in a number of more complex analogs. It is common to have different positions, varying degrees of alkylation, dealkylation or oxidation on the mother nucleus and side chains to form the chemical diversity of this family. Alkylation or dealkylation can increase or decrease the carbon number at different positions in the mother nucleus or side chain (mainly side chain), such as the number of side chains extending to C-0 to C-12; C- The 4 position may contain 2 horn methyl groups, 1 horn methyl group, no horn methyl group or methylal group; C-24 position may form methyl group or ethyl group. Different degrees of oxidation and related dehydration, reduction, decarboxylation, esterification or cyclization reactions can also occur at different positions in the mother nucleus or side chain, and then hydroxyl groups, aldehydes and ketones, carboxyl groups, A plurality of oxygen-containing functional groups such as a carboxylate, an epoxy, an ether, and a lactone. These reactions change the core skeleton of cyclopentane and polyhydrophenanthrene, causing changes such as polyhydroxy substitution, A ring aromatization, degradation, oxidative cracking of A, B, C, and D rings, and cyclization, forming a structure. More complex derivatives (Aiello, A.; Fattorusso, E.; Menna, M. Steroids from sponges: Recent reports. Steroids 1999, 64, 687-714; Sica, D.; Musumeci, D. Secosteroids of marine origin .Steroids 2004, 69, 743-756; Sarma, NS; Krishna, MS; Pasha, SG; Rao, TSP; Venkateswarlu, Y.; Parameswaran, PS Marine Metabolites: The Sterols of Soft Coral. Chemical Reviews 2009, 109, 2803 -2828). The special living environment of the ocean has caused the singularity of changes in secondary metabolites in its organisms. Many structurally specific cyclopentane and polyhydrophenanthrene derivatives have been discovered from sponges, corals and marine microorganisms. Structural changes are bound to cause changes in biological activity, and such compounds have therefore attracted widespread attention from pharmacologists.

However, among the family of compounds of cyclopentane and polyhydrophenanthrene and its derivatives, compounds having a 6/6/5/7 tetracyclic ring structure type have not been found so far.

Summary of the invention

It is an object of the present invention to provide a novel backbone compound having 6/6/5/7 tetracyclic ring or a derivative thereof.

Another object of the present invention is to provide a process for the preparation of the above 6/6/5/7 tetracyclic compound or a derivative thereof.

Still another object of the present invention is to provide the above 6/6/5/7 tetracyclic compound or a derivative thereof for the preparation of an antitumor drug, an antifungal drug, an antibacterial drug, an antiinflammatory drug, and a PTP1B inhibitor. Applications.

A first aspect of the present invention provides a compound having a tetracyclic ring structure in which each ring has a carbon number of 6, 6, 5 or 7 (i.e., A/B/C/). The carbon number of the four rings of D is 6/6/5/7, respectively, and the compound of the present invention has the following chemical structural formula:

In formula (I):

R ₁ , R ₂ , R ₃ , and R ₄ are each one or more substituents on the ring of A, B, C, and D, and R ₁ , R ₂ , R ₃ , and R ₄ are the same or different from each other, and are each independently Is hydrogen, halogen, hydroxy, peroxy, ether, amino, nitro, fluorenyl, carbonyl, nitrile, carboxyl, alkyl, cycloalkyl, aryl, aromatic heterocyclic, saturated heterocyclic, alkoxy a base, an aryloxy group, an aromatic heterocyclic oxy group or a saturated heterocyclic oxy group;

The chemical bond in the A, B, C, and D rings may be a single bond or a double bond.

The compound of formula (I) is selected from the following compounds:

1. Swinhoeisterol A: C ₂₉ H ₄₆ O ₂ , colorless powder, chemical structural formula as shown in 1;

2. Swinhoeisterol B: C ₂₉ H ₄₆ O ₃ , colorless crystal, chemical structural formula shown as 2:

3. Swinhoeisterol C: C ₂₉ H ₄₈ O ₅ , colorless powder, chemical structural formula as shown in 3;

4. Swinhoeisterol D: C ₂₉ H ₄₆ O ₆ , colorless crystal, chemical structural formula as shown in 4;

5. Swinhoeisterol E: C ₂₉ H ₄₆ O ₆ , colorless powder, chemical structural formula shown as 5.

According to a second aspect of the present invention, there is provided a process for producing the above 6/6/5/7 tetracyclic cycline compound or a derivative thereof.

The fresh sponge Theonella swinhoei collected from the South China Sea was frozen and chopped, extracted with acetone solvent, and the obtained extract was concentrated to obtain a crude extract. The crude extract was dissolved in an aqueous solution, suspended and extracted with ethyl acetate. The ethyl acetate extract is concentrated, dissolved in 90% methanol solution, extracted with petroleum ether, and the obtained extract is concentrated to obtain petroleum ether extract; the petroleum ether extract is dry-sampled and separated by silica gel column chromatography to remove petroleum ether/dichloride. Methane/acetone (from 20:1:0 to 0:1:10) was eluted by mobile phase gradient to give 19 Component (Fr. 1-19); wherein Fr 13 was separated on a Sephadex LH-20 column, eluted with petroleum ether/dichloromethane/acetone (1.5:3:1), and TLC plate was combined into 5 components ( Fr 13a-e); Fr. 13d and Fr. 13e were separately separated by preparative HPLC to obtain the compounds Swinhoeisterol A and Swinhoeisterol B; wherein Fr 14 was separated on a Sephadex LH-20 column using petroleum ether/dichloromethane/acetone (1.5). : 3:1) elution, TLC dot plates were combined into 5 fractions (Fr 14a-f); Fr. 14d was isolated by preparative HPLC to obtain compounds Swinhoeisterol C, Swinhoeisterol D and Swinhoeisterol E.

The 6/6/5/7 tetracyclic compound of the present invention or a derivative thereof can also be produced by chemical synthesis.

According to a third aspect of the present invention, there is provided a pharmaceutical use of the above 6/6/5/7 tetracyclic cycline compound or a derivative thereof.

The present invention provides the use of the above 6/6/5/7 tetracyclic compound or a derivative thereof for the preparation of an antitumor drug.

The tumor is specifically a lung cancer, a colon cancer or an osteosarcoma.

The present invention also provides the use of the above 6/6/5/7 tetracyclic compound or a derivative thereof for the preparation of an antifungal drug.

The fungus is specifically Candida albicans, Candida parapsilosis, Candida tropicalis or Cryptococcus neoformans.

The present invention also provides the use of the above 6/6/5/7 tetracyclic compound or a derivative thereof for the preparation of an antibacterial drug.

The bacteria are specifically Escherichia coli, Bacillus megaterium, Staphylococcus aureus or methicillin-resistant Staphylococcus epidermidis.

The present invention also provides the use of the above 6/6/5/7 tetracyclic paracyclic compound or a derivative thereof for preparing a protein tyrosine phosphatase 1B (PTP1B) inhibitor, which can be developed to treat type II diabetes. drug.

The present invention also provides the use of the above 6/6/5/7 tetracyclic compound or a derivative thereof for the preparation of an anti-inflammatory drug.

The activity of the anti-inflammatory drug is such that it has COX-2 inhibitory activity.

The beneficial effects of the invention are:

1. In vitro anti-tumor, in vitro antifungal, in vitro antibacterial activity, in vitro PTP1B inhibitory activity and in vitro anti-inflammatory assay, the compounds of the invention against A549 (human lung cancer cells), Lovo (human colon cancer cells) and MG63 (human osteosarcoma cells) ) and a variety of tumor cells have a significant inhibitory effect, Candida albicans, near Various fungi such as Candida or Candida tropicalis or Cryptococcus neoformans have potential antifungal activity and are potentially resistant to various bacteria such as Escherichia coli, Bacillus megaterium or Staphylococcus aureus or methicillin-resistant Staphylococcus epidermidis. Bacterial activity, potential inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), potential anti-inflammatory activity against COX-2 target inflammation, and thus useful for the preparation of anti-tumor or anti-fungal or anti-bacterial a drug or a PTP1B inhibitor or an anti-inflammatory drug;

2. The present invention provides a lead compound for the development of a new anti-tumor or anti-fungal or anti-bacterial drug or a PTP1B inhibitor or an anti-inflammatory drug, which is advantageous for the development and utilization of marine medicinal resources.

detailed description

The invention will now be further described in conjunction with specific embodiments. It is understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

Example 1: Preparation of the compounds Swinhoeisterol A to E of the present invention

3.6kg of fresh sponge Theonella swinhoei (identified by Li Jinhe, researcher from Qingdao Institute of Oceanography, Chinese Academy of Sciences) will be chopped, extracted with acetone solvent, and the obtained extract will be concentrated to obtain a crude extract. The solution was dissolved in an aqueous solution, and the mixture was suspended and extracted with ethyl acetate. The ethyl acetate extract was concentrated and dissolved in a 90% methanol solution and extracted with petroleum ether. The obtained extract was concentrated to give a petroleum ether extract (13.6 g). The petroleum ether extract was dry-packed and subjected to silica gel column chromatography, and eluted with petroleum ether/dichloromethane/acetone (from 20:1:0 to 0:1:10) as a mobile phase gradient to obtain 19 components ( Fr. 1-19).

Among them, Fr 13 (2 g) was subjected to Sephadex LH-20 column separation, eluted with petroleum ether/dichloromethane/acetone (1.5:3:1), and the TLC plate was combined to form five components (Fr 13a-e). Fr.13d and Fr.13e were separated by preparative HPLC to give Compound Swinhoeisterol A (6.0mg, 90% MeOH / H 2 O, 2.0mL / min, t R = 55.1min) and Swinhoeisterol B (2.5mg, 94% MeOH/H ₂ O, 2.0 mL/min, t _R = 22.4 min).

Among them, Fr 14 (1 g) was separated on a Sephadex LH-20 column, eluted with petroleum ether/dichloromethane/acetone (1.5:3:1), and the TLC plate was combined to form five components (Fr 14a-f). Fr. 14d was isolated by preparative HPLC to give compound Swinhoeisterol C (5.0 mg, 93% MeOH/H ₂ O, 2.0 mL/min, t _R = 30.1 min), Swinhoeisterol D (1.5 mg, 93% MeOH/H ₂ O _{, 2.0mL / min, t R =} 40.4min) and Swinhoeisterol E (2.1mg, 94% MeOH / H 2 O, 2.0mL / min, t R = 60.4min).

Example 2: Identification of the compounds of the invention

The combination of five new skeletons isolated from the South China Sea sponge Theonella swinhoei The substance, named Swinhoeisterol A-E, has the following chemical formula 1-5:

1. Swinhoeisterol A: C ₂₉ H ₄₆ O ₂ , colorless powder, chemical structural formula 1, ¹ H and ¹³ C NMR data are shown in Table 1.

2. Swinhoeisterol B: C ₂₉ H ₄₆ O ₃ , colorless crystal, chemical structural formula 2, ¹ H and ¹³ C NMR data are shown in Table 1.

3. Swinhoeisterol C: C ₂₉ H ₄₈ O ₅ , colorless powder, chemical formula 3, ¹ H and ¹³ C NMR data are shown in Table 2.

4. Swinhoeisterol D: C ₂₉ H ₄₆ O ₆ , colorless crystal, chemical structural formula 4, ¹ H and ¹³ C NMR data are shown in Table 2.

5. Swinhoeisterol E: C ₂₉ H ₄₆ O ₆ , colorless powder, chemical formula 5, ¹ H and ¹³ C NMR data are shown in Table 3.

Table ^{1. 1} H and ¹³ C NMR data for Swinhoeisterol A (Compound 1) and Swinhoeisterol B (Compound 2) ^a

^a δin ppm, in CDCl ₃ , at 500MHz for ¹ H and 125MHz for ¹³ C experiments.

Table 2. ¹ H and ¹³ C NMR data for Swinhoeisterol C (Compound 3) and Swinhoeisterol D (Compound 4) ^a

^a δin ppm, in CDCl ₃ , at 500MHz for ¹ H and 125MHz for ¹³ C experiments.

Table 3. ¹ H and ¹³ C NMR data for Swinhoeisterol E (Compound 5) ^a

^a δin ppm, in CDCl ₃ , at 500MHz for ¹ H and 125MHz for ¹³ C experiments.

Example 3: Swinhoeisterol A-E anti-tumor experiment of the compound of the invention having a 6/6/5/7 tetracyclic ring structure

First, the experimental method

The compound of the present invention was subjected to a tumor cell proliferation inhibition test by a conventional MTT method. (For the MTT method, see, for example, Lu Qiujun, editor of the New Drug Pharmacology Research Method, Chemical Industry Press, 2007: 242-243).

1. Experimental cell strains: A549 (human lung cancer cells), Lovo (human colon cancer cells), and MG63 (human osteosarcoma cells). The experimental cell line was obtained from the Institute of Cell Research of the Chinese Academy of Sciences.

2. Experimental reagents, consumables and instruments:

DMEM medium (Invitrigen); 1640 medium (Invitrigen); McCoy's 5a (Invitrigen); serum (Invitrigen); trypsin (Invitrigen); DMSO (sigma); MTT (sigma); CCK8 (Japanese colleague); Corning; pipetting (Corning); 96-well plate (Corning); CO ₂ incubator (SANYO); microplate reader (Biotek76833)

3. Experimental medication:

The compound Swinhoeisterol A-E (1-5) of the present invention

Positive control drug: Adriamycin.

4. Cell culture

A549 cell culture: human lung adenocarcinoma cells (A549) were cultured in DMEM medium containing 10% fetal bovine serum at 37 ° C under 5% CO ₂ until the cells were covered with 70% to 80% of the bottom of the culture dish. 0.25% trypsin was digested, the cell density was adjusted to 10 ⁵ /ml, and 100 μl per well was seeded in a 96-well plate, and the experiment was performed after 18-24 hours.

Lovo cell culture: human colon cancer cells (Lovo) were cultured in DMEM containing 10% fetal calf serum, and cultured at 37 ° C under 5% CO ₂ until the cells were covered with 70% to 80% of the bottom of the culture dish. 0.25% trypsin was digested, the cell density was adjusted to 10 ⁵ /ml, and 100 μl per well was seeded in a 96-well plate, and the experiment was performed after 18-24 hours.

MG63 cell culture: human osteosarcoma cells (MG63) were cultured in McCoy's 5a medium containing 10% fetal bovine serum at 37 ° C under 5% CO ₂ . When the cells reached about 10 ⁶ , centrifuge at 1000 rpm for 5 min. The cell density was 10 ⁵ /ml, and 100 μl per well was seeded in a 96-well plate, and the experiment was performed after 18 to 24 hours.

5. Cell viability assay

A549, Lovo, and MG63 cell viability assays: 96-well plates were seeded at a cell concentration of 10 ⁴ cells/well 24 h before the experiment. Each well was administered with 1 μl, and the final concentration was 30 μg/ml, respectively, and three replicate groups, a DMSO-negative control group, and a doxorubicin (30 μg/ml) positive control group were established. After administration, incubation was carried out for 24 h at 37 ° C under 5% CO ₂ . 10 μl of 5 mg/ml MTT (thiazole blue) was added to each well and incubated for 4 h at 37 ° C under 5% CO ₂ . Aspirate the cell culture medium in the culture plate. 150 μl of DMSO solution was added to each well and shaken at 37 ° C for 15 min. The OD value at 570 nm was measured with a microplate reader.

Second, the experimental results

The in vitro cytotoxic activity of the compound Swinhoeisterol AE (1-5) was determined by MTT assay. The tumor cell inhibition rate of each compound is shown in Table 1, wherein the sample concentration was 30 μg/ml, the doxorubicin concentration was 30 μg/ml, and the inhibition rate unit. for%.

Table 4 Tumor cell proliferation inhibition test of compound Swinhoeisterol A-E (1-5)

Compound	A549	Lovo	MG63
				Swinhoeisterol A	12.6	13.4	10.3
Swinhoeisterol B	29.6	19.2	20.0
				Swinhoeisterol C	9.2	9.2	10.4
Swinhoeisterol D	11.3	13.2	12.0
				Swinhoeisterol E	19.1	18.9	24.1
Adriamycin	3.08	3.56	3.17

As can be seen from Table 4, the compound Swinhoeisterol A-E (1-5) against A549 (human lung cancer cells), Both Lovo (human colon cancer cells) and MG63 (human osteosarcoma cells) have different degrees of inhibition.

Example 4: Antifungal experiment of the compound Swinhoeisterol A-E of the 6/6/5/7 tetracyclic ring structure of the present invention

First, the experimental method

The compound of the present invention was subjected to an in vitro antibacterial test by agar diffusion method (for agar diffusion method, see Lin Yong, ed., Medicinal Microbiology Foundation, Chemical Industry Press, 2006, 335-361).

1. Experimental strain

They are clinical strains of globular bacteria: Candida albicans, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, which are provided by the fungal laboratory of Changhai Hospital, and are identified by morphology and biochemistry.

All the experimental strains were activated on the sandcastle dextrose agar medium (SDA). The deep fungus was cultured at 35 ° C for one week. After the shallow fungus was cultured at 28 ° C for 2 weeks, the monoclonal clones were picked and activated again. The secondary obtained monoclonal was placed on the SDA slope, cultured by the above method, and stored at 4 ° C until use.

2, the culture solution

RPMI 1640 medium: RPMI1640 (Gibco BRL) 10g, NaHCO3 2.0g, morpholinepropanesulfonic acid (MOPS, Sigma) 34.5g (0.165M), add three distilled water 900ml dissolved, 1mol / L NaOH adjust the pH to 7.0 (25 ° C), constant volume to 1000 ml, filtered and sterilized, stored at 4 ° C. Sandcastle glucose agar medium (SDA): peptone 10g, glucose 40g, agar 18g, add three distilled water 900ml dissolved, add 2mg / ml chloramphenicol aqueous solution 50ml, adjust the pH to 7.0, constant volume to 1000ml, after autoclaving 4 save.

YEPD culture solution: yeast extract 10g, peptone 20g, glucose 20g, add three distilled water 900ml dissolved, add 2ml / ml chloramphenicol aqueous solution 50ml, dilute to 1000ml, stored at 4 ° C after autoclaving.

3, experimental medication

Positive controls: fluconazole, ketoconazole and itraconazole (ICZ) were provided by the Department of Pharmacology, Second Military Medical University.

4, test method

(1) Preparation of bacterial solution

Before the experiment, a small amount of globular bacteria such as Cryptococcus neoformans, Candida albicans and Candida parapsilosis was picked from the SDA medium preserved at 4 °C by inoculation circle, inoculated into 1 ml of YEPD culture medium, and cultured at 35 ° C, shaking at 250 rpm for 16 h. To make the fungus in the late stage of the exponential growth phase. The bacterial solution was taken to 1 ml of YEPD culture solution, and reactivated by the above method. After 16 hours, the cells were counted by a hemocytometer, and the concentration of the bacterial solution was adjusted to 1 × 10 ^{3 -} 5 × 10 ³ /ml with RPMI 1640 culture solution.

(2) Preparation of liquid medicine

The test samples were prepared into 6.4 mg/ml solution in DMSO, and stored at -20 ° C. The solution was taken out and placed in a 35 ° C incubator for dilution before the experiment.

(3) Preparation of drug sensitive plate

A sterile 96-well plate was taken, and RPMI 1640 100 ml was added to each row of wells as a blank control; freshly prepared bacterial solution 100 ml was added to each of the 3 to 12 wells; 200 ml of the bacterial solution and 2 ml of the test compound solution were added to the 2nd well. The No. 2 to No. 11 wells were diluted by 10 steps, so that the final concentrations of the drugs in each well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/ml, respectively. Less than 1%; No. 12 well contains no drug and is used as a negative control. Each of the susceptibility plates was cultured at 35 °C.

(4) MIC value determination

The strains were cultured at 35 ° C for 24 h and 72 h, respectively, and the OD values of the wells were measured at 620 nm using an enzyme labeling analyzer. The drug concentration in the lowest concentration well with an OD value of more than 80% was MIC ₈₀ compared to the negative control well.

When the MIC ₈₀ value of the sample exceeds the measured concentration range, the statistics are as follows: when the MIC ₈₀ value is higher than the highest concentration of 64 μg/ml, it is calculated as >64 μg/ml; when the MIC ₈₀ value is the lowest concentration or below the lowest concentration, No difference was made, which was <0.125 μg/ml.

The above experiments were performed in parallel 2 to 3 times. When the MIC ₈₀ value can be accurately repeated or only one concentration is accepted, it is accepted as a MIC ₈₀ value at a higher concentration; when the MIC ₈₀ values differ by more than two concentrations, Re-experiment until it meets the requirements.

Second, the experimental results

Five compounds were screened for fungal inhibitory activity. The results showed that Swinhoeisterol A-E (1-5) had different degrees of inhibition on four fungi, see Table 5.

Table 5. Screening of antifungal activity (MIC ₈₀ : μg/ml)

Example 5: Antibacterial experiment of the compound Swinhoeisterol A-E having the 6/6/5/7 tetracyclic ring structure of the present invention

experimental method

In vitro antibacterial test of the compound of the present invention by agar diffusion method

1. Test bacteria (provided by the Marine Drug Research Center of the Second Military Medical University of the People's Liberation Army)

Bacteria: Escherichia coli, Bacillus megaterium, Staphyloccocus aureus ATCC29213, methicillin-resistant Staphylococcus epidermidis (MRSE).

2. Experimental medication

1) Positive control drugs: Penicillin (purchased from Harbin Pharmaceutical Group Pharmaceutical Factory), Streptomycin (purchased from North China Pharmaceutical Co., Ltd.);

2) Negative control: Acetone (purchased from Sinopharm Chemical Reagent Co., Ltd.);

3. Experimental steps

Penicillin, streptomycin, and Swinhoeisterol AE were separately formulated into a solution having a concentration of 2 mg/ml with acetone, and a single test dose of 25 μl was used. The above bacteria were prepared into a bacterial solution by using 7 ml of sterilized water according to the requirements of aseptic operation, and the concentration was 1.0×10 ⁵ cfu/ml, and 4 ml of the bacterial liquid was respectively sprayed with a sprayer, and uniformly sprayed on the surface of the culture medium of each petri dish ( The experimental bacteria were cultured in Mueller-Hinton broth medium, and when they were grown for 8-12 h to about 0.5 Mcfarland concentration, two pieces of sterile filter paper with a diameter of about 1 cm were placed in each dish to cover the medium. On the surface, 25 μl of the above-prepared drug solution was separately dropped on the filter paper, and the petri dish lid was capped for cultivation. The corresponding culture medium type, strain, compound name, and inoculation time are indicated on the lids of each culture dish. The size (radius) of the zone of inhibition was measured on time and the test was performed 3 times in parallel. The results are shown in Table 6.

Table 6. Agar diffusion test activity screening (mm)

sample	E.coli	Bacillus megaterium	Staphylococcus aureus	Methicillin-resistant Staphylococcus epidermidis
					Swinhoeisterol A	4.0	7.5	10.5	6.5
Swinhoeisterol B	8.5	9.5	13.0	13.5
					Swinhoeisterol C	5.1	8.5	12.4	8.2
Swinhoeisterol D	11.5	14.3	10.3	15.1
					Swinhoeisterol E	13.4	12.2	17.2	11.9

penicillin	6.5	6.0	15.0	\
					Streptomycin	8.0	5.5	9.5	15.4
acetone	\	\	\	\

As can be seen from Table 6, Swinhoeisterol A-E (1-5) has different degrees of inhibition on Escherichia coli, Bacillus megaterium, Staphylococcus aureus, and methicillin-resistant Staphylococcus epidermidis.

Example 6: Inhibitory activity of the compound Swinhoeisterol A-E (1-5) having a 6/6/5/7 tetracyclic ring structure on PTP1B

PTP1B is a member of the protein tyrosine phosphatase family and is widely expressed in various tissue cells in vivo. It is used in combination with tyrosine kinase (PTK) for various protein substrates to regulate tyrosine phosphorylation. Level, which in turn regulates cellular physiological functions. PTP1B can dephosphorylate protein tyrosine and plays an important negative regulatory role in the insulin signal transduction pathway. The above studies indicate that PTP1B is expected to be a new target for the treatment of type 2 diabetes.

The activity test was carried out by a phosphatase assay: in a 0.2 mL reaction solution at 30 ° C with an appropriate concentration of p-nitrophenyl phosphate (pNPP) as a substrate. A buffer of pH=7.0 was formulated with 50 mL of 3,3-glutarate and 1 mL of EDTA. 0.15 M NaCl was used to maintain the ionic strength of the solution. The reaction was started by adding the enzyme. After 2-3 minutes, the reaction was terminated by adding 1 mL of 1 N NaOH. The non-enzymatic hydrolysis of the substrate was corrected by the enzyme-free measurement system. of. The amount of p-nitrophenol product was determined by absorption at 405 nm with a molar absorptivity of 18000 M ^-1 cm ^-1 . The Michaelis-Menten kinetic parameters were obtained by nonlinear regression of the v-pair [S] data into the Michaelis-Menten equation using the GraFit program (Erithacus software). Determination of Ki value: The initial reaction rate of 8 different concentrations of p-nitrophenyl phosphate (0.2Km-5Km) was determined using different concentrations of inhibitors, using V=VmaxS/[Km(1+Ki)+S] The equation is obtained (where the Ki value is the IC ₅₀ ). The IC ₅₀ value was obtained using a similar method.

The compounds Swinhoeisterol AE (1-5) prepared in the above examples inhibited the activity of PTP1B with IC ₅₀ values of 7.2, 18.3, 12.4, 5.3 and 15.6 μg/mL, respectively.

Example 7: Anti-inflammatory activity test of the compound Swinhoeisterol A-E (1-5) having a 6/6/5/7 tetracyclic ring structure of the present invention

To investigate the anti-inflammatory activity of the compounds, the compound Swinhoeisterol A-E (1-5) was screened for inhibition of COX-2 activity.

1. Experimental method

2',7'-dichlorodihydrofluorescein diethyl ester (DCDHF-DA), positive control drug Celecoxib purchased from Sigma; Shanghai Polarstar Plate Reader (BMG Labtechnologies, Australia); Bac-to-Bac ^TM recombination rod Viral expression system, fetal bovine serum, RPMI-1640 medium, Grace culture solution were purchased from Gibco BRL, human monocyte cell line THP-1 and insect cell Spodoptera frugiterda (sf-9) were purchased from Shanghai Biochemical Cell Institute of Chinese Academy of Sciences.

THP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and Sf-9 cells were incubated at 28 ° C in Grace culture medium containing 10% heat-inactivated fetal bovine serum. Insect sf-9 cells containing recombinant human COX-2 (hCOX-2) protein were reported in the literature (Shi Dayong, Li Xiaohong, Li Jing, Guo Shuju, Su Hua, Seaweed Extract Epoxy Plus Enzyme-2 Inhibition Activity, Marine Science , 2009, 33, 30-32) was prepared and stored in liquid nitrogen prior to use. All test compounds and control drugs were dissolved in dimethyl sulfoxide, and the final concentration of dimethyl sulfoxide was less than 0.1%, and arachidonic acid was dissolved in ethanol, and the final concentration of ethanol was less than 0.1%.

Sf-9 cells (1×10 ⁵ /mL) containing hCOX-2 protein and test compound and positive control drug

(final concentration 10 μM) was pre-incubated in 96-well plates for 30 min, then DCDHF-DA (final concentration 2.5 μM) and arachidonic acid (final concentration 2.5 μM) were added. After 10 min, the excitation wavelength was 485 nm and the emission wavelength was 520 nm. The fluorescence absorbance is measured, and the inhibition rate (IR%) is calculated as follows:

IR%=(C-Sam)/(C-B)*100

IR: Inhibitory rate; Sam: ROS production in drug treatment groups; B: Blank groups; C: Control groups

2, the experimental results

Table 7. Screening of Swinhoeisterol A-E (1-5) for inhibition of COX-2 activity

sample	Celecoxib	1	2	3	4	5
							Inhibition rate (%, 10 μM)	40.0	53.2	57.9	27.1	36.8	44.3

As can be seen from Table 7, Swinhoeisterol A-E (1-5) all have varying degrees of inhibitory activity of COX-2.

The above experimental results indicate that the compounds of the present invention have good antitumor activity or potential antifungal activity or potential antibacterial activity or potential PTP1B inhibitory activity or potential anti-inflammatory activity, and thus can be used for preparing antitumor or antifungal or Antibacterial drugs or PTP1B inhibitors or anti-inflammatory drugs, the present invention opens up new avenues for further research and development of new anti-tumor or anti-fungal or anti-bacterial drugs or PTP1B inhibitors or anti-inflammatory drugs.

The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited by the foregoing embodiments, and that the present invention is described in the foregoing description and the description of the present invention. Such changes and modifications are intended to fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and their equivalents.

Claims (13)

1. A compound having a tetracyclic ring structure, wherein each of the ring carbon atoms in the tetracyclic ring structure is 6, 6, 5 or 7, and the chemical structural formula is as shown in (I):

   

   In formula (I):

   R ₁ , R ₂ , R ₃ , and R ₄ are each one or more substituents on the ring of A, B, C, and D, and R ₁ , R ₂ , R ₃ , and R ₄ are the same or different from each other, and are each independently Is hydrogen, halogen, hydroxy, peroxy, ether, amino, nitro, fluorenyl, carbonyl, nitrile, carboxyl, alkyl, cycloalkyl, aryl, aromatic heterocyclic, saturated heterocyclic, alkoxy A aryl group, an aryloxy group, an aromatic heterocyclic oxy group or a saturated heterocyclic oxy group.
2. A compound having a tetracyclic ring structure according to claim 1, wherein the compound has a chemical structural formula such as 1, 2, 3, 4 or 5:

   
3. A method for preparing a compound having a tetracyclic ring structure according to claim 2, wherein the method comprises the steps of:

   The fresh sponge Theonella swinhoei collected from the South China Sea was frozen and chopped, extracted with acetone solvent, and the obtained extract was concentrated to obtain a crude extract. The crude extract was dissolved in an aqueous solution, suspended and extracted with ethyl acetate. The ethyl acetate extract is concentrated, dissolved in 90% methanol solution, extracted with petroleum ether, and the obtained extract is concentrated to obtain petroleum ether extract; the petroleum ether extract is dry-sampled and separated by silica gel column chromatography to remove petroleum ether/dichloride. The methane/acetone was eluted from a mobile phase gradient of 20:1:0 to 0:1:10 to obtain 19 components of Fr.1-19; wherein Fr 13 was separated by Sephadex LH-20 column, and petroleum ether/dichlorobenzene was used. Methane/acetone 1.5:3:1 elution, TLC dot plates were combined into 5 components Fr.13a-e; Fr.13d and Fr.13e were separated by preparative HPLC, respectively, to obtain chemical structural formulas as shown in 1 and 2. Compound; wherein Fr 14 was separated by Sephadex LH-20 column, eluted with petroleum ether/dichloromethane/acetone 1.5:3:1, TLC plate was combined to form 5 components Fr.14a-f; Fr.14d was prepared. The HPLC separation is carried out to obtain compounds of the chemical formulas such as 3, 4 and 5.
4. Use of a compound having a tetracyclic ring structure according to claim 1 or 2 for the preparation of an antitumor drug.
5. The compound having a tetracyclic ring structure according to claim 4, which is in the preparation of an antitumor drug, characterized in that the tumor is lung cancer, colon cancer or osteosarcoma.
6. Use of a compound having a tetracyclic ring structure according to claim 1 or 2 for the preparation of an antifungal drug.
7. The use of a compound having a tetracyclic ring structure according to claim 6 for the preparation of an antifungal drug, characterized in that the fungus is Candida albicans, Candida parapsilosis, Candida tropicalis or Cryptococcus neoformans.
8. Use of a compound having a tetracyclic ring structure according to claim 1 or 2 for the preparation of an antibacterial drug.
9. The use of a compound having a tetracyclic ring structure according to claim 8 for the preparation of an antibacterial drug, characterized in that the bacterium is Escherichia coli, Bacillus megaterium, Staphylococcus aureus or methicillin-resistant epidermis staphylococcus.
10. Use of a compound having a tetracyclic ring structure according to claim 1 or 2 for the preparation of a protein tyrosine phosphatase 1B inhibitor.
11. A compound having a tetracyclic ring structure according to claim 1 or 2 for use in the preparation of a medicament for the treatment of type II diabetes.
12. Use of a compound having a tetracyclic ring structure according to claim 1 or 2 for the preparation of an anti-inflammatory drug.
13. The use of a compound having a tetracyclic ring structure according to claim 12 for the preparation of an anti-inflammatory drug, characterized in that the activity of the anti-inflammatory drug is COX-2 inhibitory activity.