Cyclic dipeptide compound as well as preparation method and application thereof in pharmacy

Abstract

The invention belongs to the technical field of natural medicine development, and relates to preparation of I-IV cyclic dipeptide compounds and application of the I-IV cyclic dipeptide compounds in antibacterial medicine development. According to the invention, cyclic dipeptide compounds with antibacterial activity are separated from a crude extract of fungus Hormonema dematioids, and the cyclic dipeptide compounds are respectively a compound cyclic (D-isoleucine-D-valine) dipeptide (I), a compound cyclic (D-alanine-D-valine) dipeptide (II), a compound cyclic (L-proline-glycine) dipeptide (III) and a compound cyclic (D-alanine-D-phenylalanine) dipeptide (IV). An antibacterial test proves that the compound has a remarkable inhibiting effect on human pathogenic bacteria staphylococcus aureus, and the cyclic dipeptide compound can be used as a pharmaceutical active component for preparing antibacterial drugs, especially drugs for treating diseases caused by staphylococcus aureus infection. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of natural drug development, and relates to cyclic dipeptide compounds and preparation methods and pharmaceutical uses thereof, and specifically to cyclic dipeptide compounds in the fungus Hormonema dematioides and preparation methods thereof and uses thereof in preparing antibacterial drugs. Background Art

[0002] Studies have confirmed that bacteria are pathogens of several diseases, and their infection can cause various inflammations and even shock and death, posing a huge threat to human life and health. With the advent of penicillin, antibiotics have developed rapidly and have become a powerful weapon for treating bacterial infections. Clinical basic and practical research shows that with the emergence of pathogenic bacteria's resistance to antibiotics and the increasing degree of resistance, the industry urgently needs to develop more new and more effective antibacterial drugs. It is reported that there are currently three main types of development of new antibacterial drugs, including the discovery of new antibacterial lead compounds from natural resources such as microorganisms, animals and plants, the optimization design and structural modification of existing antibacterial drugs, and the combination of existing antibacterial drugs (including antibacterial enhancers); among them, the discovery of new antibacterial lead compounds from natural resources such as microorganisms, animals and plants is considered to be the most effective method, and it is also the focus and hot spot of current pharmaceutical industry and research.

[0003] The fungus Hormonema dematioides is derived from the genus Hormonema. Studies have shown that the genus Hormonema contains triterpenoid glycosides with good antifungal activity, which has good research prospects. Based on the foundation and current status of the prior art, the inventors of this application intend to provide cyclic dipeptide compounds in the fungus Hormonema dematioides, preparation methods thereof, and use thereof in the preparation of antibacterial drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a natural product with antibacterial activity and a preparation method thereof based on the prior art, and specifically to provide a cyclic dipeptide compound in the fungus Hormonema dematioides and a preparation method thereof and use thereof in the preparation of antibacterial drugs.

[0005] The invention provides cyclic dipeptide compounds in the fungus Hormonema dematioides, which include compounds cyclic (D-isoleucine-D-valine) dipeptide (I), cyclic (D-alanine-D-valine) dipeptide (II), cyclic (L-proline-glycine) dipeptide (III), and cyclic (D-alanine-D-phenylalanine) dipeptide (IV).

[0006] The present invention further provides a preparation method of the cyclic dipeptide compound in the fungus Hormonema dematioides and scientific data of its antibacterial activity.

[0007] The present invention uses modern pharmacological screening methods to evaluate the antibacterial activity of the isolated monomer compounds. Active dipeptide compounds are isolated from the crude extract of the fungus Hormonema dematioides and their minimum inhibitory concentration (MIC) values ​​are further obtained, providing reliable data support for the development of new antibacterial drugs.

[0008] More specifically, the active cyclic dipeptide compounds of the present invention have chemical structures of Formulas I to IV:

[0009]

[0010] The cyclic dipeptide compounds of the present invention are prepared by the following method:

[0011] The culture fluid of the fungus Hormonema dematioides was extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol solvents, and the crude petroleum ether extract, ethyl acetate fraction, n-butanol fraction and water-soluble extract were obtained by concentration and drying respectively; the ethyl acetate extract fraction was separated by silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate system in sequence to obtain 6 fractions (Fr.1-Fr.6), among which fraction Fr.6 was further purified by high performance liquid column chromatography (methanol-water as eluent) to separate compound I ring (D-isoleucine-D-valine) dipeptide. The n-butanol fraction was separated by reverse phase silica gel column chromatography, and gradient eluted with methanol-water as mobile phase in sequence to obtain 5 fractions (Fr.1-Fr.5), among which fraction Fr.2 was further purified by high performance liquid column chromatography (methanol-water as eluent) to separate compound II ring (D-alanine-D-valine) dipeptide and compound IV ring (D-alanine-D-phenylalanine) dipeptide. The water-soluble extract was separated by reverse-phase silica gel column chromatography and gradient elution was performed in sequence with methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5), among which fraction Fr.1 was further purified by high-performance liquid column chromatography (methanol-water as eluent) to separate compound III cyclo (L-proline-glycine) dipeptide.

[0012] In the above preparation method, the culture solution of the fungus Hormonema dematioides is extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol solvents in sequence, and concentrated and dried to obtain 12g of petroleum ether crude extract, 23g of ethyl acetate fraction, 30g of n-butanol fraction and 56g of water-soluble extract;

[0013] In the above preparation method, the ethyl acetate extraction part is separated by silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate system (50:1, 20:1, 10:1, 5:1, 1:1, 0:1) in sequence to obtain 6 fractions (Fr.1 to Fr.6), wherein fraction Fr.6 is further purified by high performance liquid column chromatography (methanol-water as eluent) to separate compound I cyclo(D-isoleucine-D-valine) dipeptide (t R =17min,3.2mg);

[0014] In the above preparation method, the n-butanol part is separated by reverse phase silica gel column chromatography, and gradient elution (1:9, 3:7, 1:1, 7:3, 0:1) is carried out in sequence with methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5), wherein the fraction Fr.2 is further purified by high performance liquid column chromatography (methanol-water as the eluent) to separate compound II cyclo(D-alanine-D-valine) dipeptide (t R =15min, 5.8mg) and compound IV cyclo(D-alanine-D-phenylalanine) dipeptide (t R =11min,4.0mg);

[0015] In the above preparation method, the water-soluble extract is separated by reverse phase silica gel column chromatography, and gradient elution (1:9, 3:7, 1:1, 7:3, 0:1) is carried out in sequence with methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5), wherein fraction Fr.1 is further purified by high performance liquid column chromatography (methanol-water as the eluent) to separate compound III cyclo(L-proline-glycine) dipeptide (t R =8min,4.3mg).

[0016] In the present invention, identification was carried out, and the results showed that compound I is cyclo(D-isoleucine-D-valine) dipeptide [cyclo(D-Ile-D-Val)]: white powder, molecular formula: C 11 H 20 N 2 O 2 ; Molecular weight: 212; 1 H-NMR (400MHz, CD 3 OD):δ H3.90(1H,dd,J=3.9,1.8Hz,H-3),3.83(1H,dd,J=3.9,1.8Hz,H-3′),2.30(1H,m,H-4),1.99(2H,d,J=6.9Hz,H-5),1.4 3(1H,m,H-4′),1.08(3H,d,J=6.8Hz,H-6′),1.00(3H,m,H-6),0.91(3H,d,J=6.9Hz,H-5′),0.89(3H,t,J=7.2Hz,H-7); 13 C-NMR (100MHz, CD 3 OD):δ C 168.6(C-1,1′),61.1(C-3),60.7(C-3′),40.1(C-4),33.1(C-4′),25.9(C-5),19.3(C-6′),17.7(C-5′),15.6(C-6),12.1(C-7).

[0017] Compound II is a cyclo(D-alanine-D-valine) dipeptide [cyclo(D-Val-D-Ala)]: colorless needle (MeOH), molecular formula: C 8 H 14 N 2 O 2 ;Molecular weight: 170; 1 H-NMR (400MHz, CD 3 OD):δ H 4.03(1H,m,H-3′),3.84(1H,m,H-3),2.29(1H,m,H-4),1.45(3H,d,J=6.9Hz,H-4′),1.05(3H,d,J=6.9Hz,H-5),0.95(3H,d,J=6.9Hz,H-6); 13 C-NMR (100MHz, CD 3 OD):δ C 171.4(C-1),169.3(C-1′),61.4(C-3),51.7(C-3′),33.3(C-4),21.1(C-4′),19.1(C-5),17.3(C-6).

[0018] Compound III is cyclo(L-proline-glycine) dipeptide [cyclo(L-Pro-Gly)]: white powder, molecular formula: C 7 H 10 N 2 O 2 ; Molecular weight: 154; 1H-NMR (400MHz, CD 3 OD):δ H 4.23(2H,m,H-3′),4.10(1H,d,J=6.8Hz,H-6),3.81(2H,m,H-5),2.33(1H,m,H-3),2.10~1.80(3H,m,H-3,4); 13 C-NMR (100MHz, CD 3 OD):δ C 172.0(C-1),166.5(C-1′),59.8(C-6),50.0(C-3′),46.3(C-5),29.4(C-3),23.3(C-4).

[0019] Compound IV is cyclo(D-alanine-D-phenylalanine) dipeptide [(cyclo(D-Ala-D-Phe)]: white powder, molecular formula: C 12 H 14 N 2 O 2 ; Molecular weight: 218; 1 H-NMR (400MHz, CD 3 OD):δ H 7.29~7.20(5H,m,H-6′~10′),4.16(1H,m,H-3),3.61(1H,m,H-3′),3.31(1H,dd,J= 13.4, 3.8Hz, H-4′), 2.99 (1H, dd, J=13.4, 5.0Hz, H-4′), 0.49 (3H, d, J=7.0Hz, H-4); 13 C-NMR (100MHz, CD 3 OD):δ C 167.6(C-1),165.7(C-1′),136.0(C-5′),129.6(C-6′,10′),128.4(C-7 ′,9′),126.5(C-8′),57.9(C-3),50.4(C-3′),40.7(C-4′),19.6(C-4).

[0020] The cyclic dipeptide compounds prepared by the present invention were subjected to an in vitro antibacterial activity test, and the activity test was determined by a broth dilution method. The results showed that the compounds had a significant inhibitory effect on Staphylococcus aureus, a human pathogenic bacterium, and the MIC values ​​were: 0.39 μg / mL of cyclic (D-isoleucine-D-valine) dipeptide, 0.01 μg / mL of cyclic (D-alanine-D-valine) dipeptide, 0.04 μg / mL of cyclic (L-proline-glycine) dipeptide, and 0.10 μg / mL of cyclic (D-alanine-D-phenylalanine) dipeptide (as shown in Table 1).

[0021] Table 1. Inhibitory effects of compounds I to IV on Staphylococcus aureus (Mean±SD, n=3)

[0022]

[0023] The present invention isolates a cyclic dipeptide compound with antibacterial activity from a crude extract of the fungus Hormonema dematioides, and further broth dilution method antibacterial test confirms that the compound has a significant inhibitory effect on human pathogenic bacteria Staphylococcus aureus. The cyclic dipeptide compound of the present invention can be used as a pharmaceutical active ingredient to prepare an antibacterial drug, and further used to treat diseases caused by Staphylococcus aureus infection. DETAILED DESCRIPTION

[0024] Example 1. Preparation of cyclic dipeptide compounds

[0025] The culture fluid of the fungus Hormonema dematioides was extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol solvents, and concentrated and dried to obtain 12 g of petroleum ether crude extract, 23 g of ethyl acetate part, 30 g of n-butanol part and 56 g of water-soluble extract; the ethyl acetate extract part was separated by silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate system (50:1, 20:1, 10:1, 5:1, 1:1, 0:1) in sequence to obtain 6 fractions (Fr.1 to Fr.6), among which fraction Fr.6 was further purified by high performance liquid column chromatography (methanol-water as eluent) to separate compound I cyclo(D-isoleucine-D-valine) dipeptide (t R =17min, 3.2mg). The n-butanol fraction was separated by reverse phase silica gel column chromatography, and gradient elution (1:9, 3:7, 1:1, 7:3, 0:1) was performed in sequence with methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5), of which fraction Fr.2 was further purified by high performance liquid column chromatography (methanol-water as the eluent) to separate compound II cyclo(D-alanine-D-valine) dipeptide (t R=15min, 5.8mg) and compound IV cyclo(D-alanine-D-phenylalanine) dipeptide (t R =11min, 4.0mg). The water-soluble extract was separated by reverse phase silica gel column chromatography, and gradient elution (1:9, 3:7, 1:1, 7:3, 0:1) was performed in sequence with methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5), of which fraction Fr.1 was further purified by high performance liquid column chromatography (methanol-water as the eluent) to separate compound III cyclo(L-proline-glycine) dipeptide (t R =8min,4.3mg).

[0026] Compound I is cyclo(D-isoleucine-D-valine) dipeptide [cyclo(D-Ile-D-Val)]: white powder, molecular formula: C 11 H 20 N 2 O 2 ; Molecular weight: 212; 1 H-NMR (400MHz, CD 3 OD):δ H 3.90(1H,dd,J=3.9,1.8Hz,H-3),3.83(1H,dd,J=3.9,1.8Hz,H-3′),2.30(1H,m,H-4),1.99(2H,d,J=6.9Hz,H-5),1.4 3(1H,m,H-4′),1.08(3H,d,J=6.8Hz,H-6′),1.00(3H,m,H-6),0.91(3H,d,J=6.9Hz,H-5′),0.89(3H,t,J=7.2Hz,H-7); 13 C-NMR (100MHz, CD 3 OD):δ C 168.6(C-1,1′),61.1(C-3),60.7(C-3′),40.1(C-4),33.1(C-4′),25.9(C-5),19.3(C-6′),17.7(C-5′),15.6(C-6),12.1(C-7).

[0027] Compound II is a cyclo(D-alanine-D-valine) dipeptide [cyclo(D-Val-D-Ala)]: colorless needle (MeOH), molecular formula: C 8 H 14 N 2 O 2 ;Molecular weight: 170; 1 H-NMR (400MHz, CD 3 OD):δ H4.03(1H,m,H-3′),3.84(1H,m,H-3),2.29(1H,m,H-4),1.45(3H,d,J=6.9Hz,H-4′),1.05(3H,d,J=6.9Hz,H-5),0.95(3H,d,J=6.9Hz,H-6); 13 C-NMR (100MHz, CD 3 OD):δ C 171.4(C-1),169.3(C-1′),61.4(C-3),51.7(C-3′),33.3(C-4),21.1(C-4′),19.1(C-5),17.3(C-6).

[0028] Compound III is cyclo(L-proline-glycine) dipeptide [cyclo(L-Pro-Gly)]: white powder, molecular formula: C 7 H 10 N 2 O 2 ; Molecular weight: 154; 1 H-NMR (400MHz, CD 3 OD):δ H 4.23(2H,m,H-3′),4.10(1H,d,J=6.8Hz,H-6),3.81(2H,m,H-5),2.33(1H,m,H-3),2.10-1.80(3H,m,H-3,4); 13 C-NMR (100MHz, CD 3 OD):δ C 172.0(C-1),166.5(C-1′),59.8(C-6),50.0(C-3′),46.3(C-5),29.4(C-3),23.3(C-4).

[0029] Compound IV is cyclo(D-alanine-D-phenylalanine) dipeptide [cyclo(D-Ala-D-Phe)]: white powder, molecular formula: C 12 H 14 N 2 O 2 ; Molecular weight: 218; 1 H-NMR (400MHz, CD 3 OD):δ H7.29~7.20(5H,m,H-6′~10′),4.16(1H,m,H-3),3.61(1H,m,H-3′),3.31(1H,dd,J= 13.4, 3.8Hz, H-4′), 2.99 (1H, dd, J=13.4, 5.0Hz, H-4′), 0.49 (3H, d, J=7.0Hz, H-4); 13 C-NMR (100MHz, CD 3 OD):δ C 167.6(C-1),165.7(C-1′),136.0(C-5′),129.6(C-6′,10′),128.4(C-7 ′,9′),126.5(C-8′),57.9(C-3),50.4(C-3′),40.7(C-4′),19.6(C-4).

[0030] Example 2. In vitro antibacterial activity test using the inhibition zone method

[0031] Staphylococcus aureus was cultured at 28°C and 150rpm shaking conditions, and evenly smeared on agar medium (1L sterile water contains 5g peptone, 3g beef extract, 5g sodium chloride and 15g agar). 10μL of the cyclic dipeptide compound solution was applied to a sterile paper disc (6mm in diameter), cultured at 28°C for 1 day with the culture medium containing the microbial solution, and the growth of Staphylococcus aureus was observed. If the test compound diffused from the paper disc is sensitive to Staphylococcus aureus, an inhibition zone around the circular paper disc can be observed. Ciprofloxacin was used as a positive control for this test, and each test was repeated 3 times.

[0032] Example 3. Broth dilution method antibacterial activity test

[0033] Add 200 μL of cyclodipeptide compound solution with an initial concentration of 1 mg / mL to a 96-well plate, and then serially dilute the solution to twice the final concentration. Transfer 100 μL of bacterial solution to each well containing the compound solution. The bacterial solution concentration is 1×10 6 CFU / mL, culture at 28℃ for 15 hours, and observe the turbidity of the bacterial solution in the 96-well plate. If the transparency is high, it means that the growth of bacteria is inhibited. The lowest concentration of the compound in the well is the MIC. In this experiment, each well is set up with a bacterial solution without drug and a sterile culture medium blank control, and each test is performed 3 times.

[0034] The reagents used in the experiments of the present invention are all well-known in the art and can be purchased commercially.

Claims

1. Active cyclic dipeptide compounds of formula I to IV:

2. The method for preparing the active cyclic dipeptide compounds of formula I to IV according to claim 1, characterized in that: include, The culture fluid of the fungus Hormonema dematioides is extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol solvents, and concentrated and dried to obtain a petroleum ether crude extract, an ethyl acetate part, an n-butanol part and a water-soluble extract; the ethyl acetate extraction part is separated by silica gel column chromatography, and gradient eluted with a petroleum ether-ethyl acetate system in sequence to obtain 6 fractions Fr.1 to Fr.6, wherein the fraction Fr.6 is further purified by high performance liquid column chromatography (methanol-water as an eluent) to separate and obtain compound I ring (D-isoleucine-D-valine) dipeptide; the n-butanol part is separated by reverse phase silica gel column chromatography, and gradient eluted with methanol-water as a mobile phase in sequence to obtain 5 fractions Fr.1 to Fr.5, wherein the fraction Fr.2 is further purified by high performance liquid column chromatography (methanol-water as an eluent) to separate and obtain compound II ring (D-alanine-D-valine) dipeptide and compound IV ring (D-alanine-D-phenylalanine) dipeptide. The water-soluble extract was separated by reverse-phase silica gel column chromatography and gradient elution was performed in sequence with methanol-water as the mobile phase to obtain 5 fractions Fr.1 to Fr.5, among which fraction Fr.1 was further purified by high-performance liquid column chromatography (methanol-water as eluent) to separate compound III ring (L-proline-glycine) dipeptide.

3. The preparation method according to claim 2, characterized in that: in, The ethyl acetate extraction part was separated by silica gel column chromatography and gradient eluted with petroleum ether-ethyl acetate system (50:1, 20:1, 10:1, 5:1, 1:1, 0:1) to obtain 6 fractions (Fr.1-Fr.6).

4. The preparation method according to claim 2, characterized in that: in, The n-butanol fraction was separated by reverse phase silica gel column chromatography, and gradient elution was performed in sequence (1:9, 3:7, 1:1, 7:3, 0:1) using methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5).

5. The preparation method according to claim 2, characterized in that: in, The water-soluble extract was separated by reverse phase silica gel column chromatography, and gradient elution was performed in sequence (1:9, 3:7, 1:1, 7:3, 0:1) using methanol-water as the mobile phase to obtain 5 fractions (Fr.1 to Fr.5).

6. Use of the active cyclic dipeptide compounds of formula I to IV according to claim 1 in the preparation of antibacterial drugs.

7. Use of the active cyclic dipeptide compounds of formula I to IV according to claim 1 in the preparation of drugs for treating infectious diseases caused by Staphylococcus aureus.

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