Alkyne compounds and application thereof in preparation of antibacterial drugs

Abstract

The invention belongs to the field of natural medicines and chemical medicines, and discloses alkyne compounds and application thereof in preparation of antibacterial medicines. The alkyne compound is named as chienhainanins G-L, and the structure of the alkyne compound is as shown in 1-6. The alkyne compound and the composition containing the alkyne compound have remarkable antibacterial activity on enterococcus faecalis and methicillin-resistant staphylococcus aureus at lower concentration, and can be used for preparing antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of natural medicine and chemical medicine, and particularly relates to a new class of acetylene compounds and the application thereof in preparing antibacterial drugs. BACKGROUND

[0002] The widespread use and even abuse of antibacterial drugs has made bacterial drug resistance increasingly serious, and drug-resistant bacterial infection has become a major threat to global public health. The World Health Organization has listed antimicrobial drug resistance as one of the 10 threats to global health in 2019. However, the current development speed of antibacterial drugs is seriously lagging behind the development speed of bacterial drug resistance, and the current situation of clinical antibacterial drugs for drug-resistant bacterial infection is not optimistic. In order to cope with the challenge of bacterial drug resistance and curb the development and spread of bacterial drug resistance, the WHO announced 12 drug-resistant bacteria with almost no drugs in the world in 2017, called for the development of antibacterial drugs against these drug-resistant bacteria, and China also issued the National Action Plan for Controlling Bacterial Drug Resistance (2016-2020) and the National Action Plan for Controlling Microbial Drug Resistance (2022-2025) in 2016 and 2022, respectively, encouraging the development of new antibacterial drugs for drug-resistant bacterial infection. Therefore, it is of great significance to develop new antibacterial drugs for drug-resistant bacteria.

[0003] Chieniodendron hainanense (Merr.) Tsiang & P.T.Li is the only plant of Chieniodendron in Annonaceae, which is produced in Hainan, Guangdong and Guangxi, etc. of China. At present, the chemical composition and pharmacological activity of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li are still blank. Therefore, the research on the chemical composition and pharmacological activity of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li can provide help and guidance for the use of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li. SUMMARY

[0004] The primary purpose of the present application is to provide a new class of acetylene compounds or pharmaceutically acceptable salts thereof.

[0005] The acetylene compounds or pharmaceutically acceptable salts thereof are obtained by extraction, separation and purification from Chieniodendron hainanense (Merr.) Tsiang & P.T.Li.

[0006] The second purpose of the present application is to provide a preparation method of the acetylene compounds or pharmaceutically acceptable salts thereof.

[0007] The third purpose of the present application is to provide the application of the acetylene compounds in preparing antibacterial drugs.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] A class of acetylene compounds, named chienhainanins G-L, has the structure as shown in formula 1-6.

[0010]

[0011] Further, the present application provides a preparation method of the acetylenic compound, comprising the following steps:

[0012] The branches and leaves of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li are crushed into powder, and then extracted by percolation with 95% (w / w) ethanol. The extract is concentrated and then extracted with dichloromethane. The dichloromethane extract is separated and purified by silica gel column chromatography, octadecyl-bonded silica gel (ODS) column chromatography, Sephadex LH-20 and preparative HPLC to obtain the acetylenic compound.

[0013] Specifically, the preparation method of the acetylenic compound comprises the following steps:

[0014] (1) The branches and leaves of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li are crushed into powder, and then extracted by percolation with 90-95% (w / w) ethanol. The percolation extracts are combined and concentrated under reduced pressure until no alcohol smell is left to obtain a total extract.

[0015] (2) The total extract is mixed with 100-200 mesh silica gel and then extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract, respectively.

[0016] (3) The dichloromethane extract is subjected to silica gel column chromatography, and gradient elution is performed with petroleum ether-ethyl acetate (100:1-0:100) as the eluent. Similar fractions are combined according to thin layer chromatography (TLC) analysis to obtain 24 main fractions Fr.1-Fr.24.

[0017] (4) Fractions Fr.15 (petroleum ether / ethyl acetate 100:20 elution part) and Fr.16 (petroleum ether / ethyl acetate 100:20 elution part) are combined and then subjected to silica gel column chromatography. Gradient elution is performed with petroleum ether-ethyl acetate (15:1-1:1) as the eluent to obtain 6 sub-fractions Fr.15A-Fr.15F.

[0018] (5) Fraction Fr.15D (petroleum ether / ethyl acetate 12:1-7:1 elution part) is subjected to octadecyl-bonded silica gel (ODS) column chromatography. Gradient elution is performed with methanol-water (40:60-100:0) as the eluent to obtain 20 fractions Fr.15Da-Fr.15Dt.

[0019] (6) The fraction Fr.15Do (the methanol-water 70:30 elution part) is separated and purified by reverse phase preparative HPLC, eluted with acetonitrile-water in a volume ratio of 50-60:50-40 to obtain 7 fractions Fr.15Do1-Fr.15Do7;

[0020] (7) The fraction Fr.15Dp (the methanol-water 75:25 elution part) is separated and purified by reverse phase preparative HPLC, eluted with acetonitrile-water in a volume ratio of 50-60:50-40 to obtain 7 fractions Fr.15Dp1-Fr.15Dp7.

[0021] (8) The fraction Fr.15Do1 is separated and purified by reverse phase preparative HPLC, eluted with methanol-water in a volume ratio of 75:25 to obtain chienhainanin G (compound 1) and chienhainanin L (compound 6); the fraction Fr.15Dp1 is separated and purified by reverse phase preparative HPLC, eluted with acetonitrile-water in a volume ratio of 55:45 to obtain chienhainanin K (compound 5); the fraction Fr.15Dp2 is separated and purified by reverse phase preparative HPLC, eluted with methanol-water in a volume ratio of 75:25 to obtain chienhainanin H (compound 2); the fraction Fr.15Dp3 is separated and purified by reverse phase preparative HPLC, eluted with acetonitrile-water in a volume ratio of 60:40 to obtain chienhainanin J (compound 4); the fraction Fr.15Dp4 is separated and purified by reverse phase preparative HPLC, eluted with acetonitrile-water in a volume ratio of 60:40 to obtain chienhainanin I (compound 3).

[0022] The application provides a pharmaceutical composition containing the acetylenic compound and a pharmaceutically acceptable carrier or excipient thereof.

[0023] The application provides application of the acetylenic compound in preparation of an antibacterial drug.

[0024] The application provides application of the pharmaceutical composition containing the acetylenic compound in preparation of an antibacterial drug.

[0025] The bacteria are one of methicillin-resistant Staphylococcus aureus or Enterococcus faecalis.

[0026] The application has the following advantages and effects relative to the prior art:

[0027] (1) The application finds a kind of novel acetylenic compound chienhainanins G-L in Broussonetia cumingsii, which is a kind of novel chemical entity.

[0028] (2) The present application finds that chienhainanins G-L have significant antibacterial activity against methicillin-resistant Staphylococcus aureus and Enterococcus faecalis at a lower concentration (2-16 μg / mL). BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 H NMR spectrum of the acetylenic compound chienhainanin G (Compound 1). 1 H NMR spectrum of the acetylenic compound chienhainanin G (Compound 1).

[0030] Figure 2 C NMR spectrum of the acetylenic compound chienhainanin G (Compound 1). 13 C NMR spectrum of the acetylenic compound chienhainanin G (Compound 1).

[0031] Figure 3 H NMR spectrum of the acetylenic compound chienhainanin H (Compound 2). 1 H NMR spectrum of the acetylenic compound chienhainanin H (Compound 2).

[0032] Figure 4 C NMR spectrum of the acetylenic compound chienhainanin H (Compound 2). 13 C NMR spectrum of the acetylenic compound chienhainanin H (Compound 2).

[0033] Figure 5 H NMR spectrum of the acetylenic compound chienhainanin I (Compound 3). 1 H NMR spectrum of the acetylenic compound chienhainanin I (Compound 3).

[0034] Figure 6 C NMR spectrum of the acetylenic compound chienhainanin I (Compound 3). 13 C NMR spectrum of the acetylenic compound chienhainanin I (Compound 3).

[0035] Figure 7 H NMR spectrum of the acetylenic compound chienhainanin J (Compound 4). 1 H NMR spectrum of the acetylenic compound chienhainanin J (Compound 4).

[0036] Figure 8 C NMR spectrum of the acetylenic compound chienhainanin J (Compound 4). 13 C NMR spectrum of the acetylenic compound chienhainanin J (Compound 4).

[0037] Figure 9 H NMR spectrum of the acetylenic compound chienhainanin K (Compound 5). 1 H NMR spectrum of the acetylenic compound chienhainanin K (Compound 5).

[0038] Figure 10 C NMR spectrum of the acetylenic compound chienhainanin K (Compound 5). 13 C NMR spectrum of the acetylenic compound chienhainanin K (Compound 5).

[0039] Figure 11 Chienhainanin L (Compound 6) is a cinnamic acid derivative compound 1 H NMR spectrum.

[0040] Figure 12 Chienhainanin L (Compound 6) is a cinnamic acid derivative compound 13 C NMR spectrum. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with the examples and the accompanying drawings, but the embodiments of the present application are not limited thereto.

[0042] Extraction, separation and structural identification of the compound of Example 1:

[0043] (1) 53 kg of Chieniodendron hainanense (Merr.) Tsiang & P.T.Li branches and leaves were crushed into coarse powder, and extracted by percolation with 80 L of 95% (w / w) ethanol. The percolation liquid was combined and concentrated under reduced pressure until no alcohol smell was left, and about 5.5 kg of total extract was obtained. The extract was mixed with 100-200 mesh silica gel, and then extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, to obtain 350 g of petroleum ether extract, 549 g of dichloromethane extract and 574 g of ethyl acetate extract.

[0044] (2) The dichloromethane extraction part was subjected to silica gel column chromatography, petroleum ether-ethyl acetate was used as the eluent, and elution gradient of petroleum ether and ethyl acetate volume ratio of 100:1, 100:2, 100:5, 100:10, 100:20, 100:100 and 0:100 was used for elution. Similar fractions were combined by thin layer chromatography (TLC) analysis to obtain 24 main fractions Fr.1-Fr.24. The fractions Fr.15 (petroleum ether / ethyl acetate 100:20 elution part, 38 g) and Fr.16 (petroleum ether / ethyl acetate 100:20 elution part, 57 g) were combined and then subjected to silica gel column chromatography. Petroleum ether-ethyl acetate was used as the eluent, and elution gradient of petroleum ether and ethyl acetate volume ratio of 15:1, 12:1, 10:1, 8:1, 7:1, 6:1, 5:1, 2:1, 1:1 was used for elution to obtain 6 sub-fractions Fr.15A-Fr.15F. The fraction Fr.15D (petroleum ether / ethyl acetate 12:1-7:1 elution part, 40 g) was further subjected to octadecyl-bonded silica gel (ODS) column chromatography. Methanol-water was used as the eluent, and elution gradient of methanol and water volume ratio of 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 100:0 was used for elution to obtain 20 fractions Fr.15Da-Fr.15Dt. The fraction Fr.15Do (methanol-water 70:30 elution part) was separated and purified by reverse phase preparative HPLC with acetonitrile-water (volume ratio 55:45) as the eluent at a flow rate of 16 mL / min to obtain 7 fractions Fr.15Do1-Fr.15Do7. The fraction Fr.15Dp (methanol-water 75:25 elution part) was separated and purified by reverse phase preparative HPLC with acetonitrile-water (volume ratio 55:45) as the eluent at a flow rate of 16 mL / min to obtain 7 fractions Fr.15Dp1-Fr.15Dp7.

[0045] (3) The fraction Fr. 15Dol was separated and purified by reverse phase preparative HPLC with methanol-water (75:25, by volume) as eluent at a flow rate of 4 mL / min to give chienhainanin G (7.3 mg) and chienhainanin L (4.5 mg) successively; the fraction Fr. 15Dpl was separated and purified by reverse phase preparative HPLC with acetonitrile-water (55:45, by volume) as eluent at a flow rate of 16 mL / min to give chienhainanin K (7.9 mg); the fraction Fr. 15Dp2 was separated and purified by reverse phase preparative HPLC with methanol-water (75:25, by volume) as eluent at a flow rate of 4 mL / min to give chienhainanin H (5.1 mg); the fraction Fr. 15Dp3 was separated and purified by reverse phase preparative HPLC with acetonitrile-water (60:40, by volume) as eluent at a flow rate of 16 mL / min to give chienhainanin J (11.4 mg); the fraction Fr. 15Dp4 was separated and purified by reverse phase preparative HPLC with acetonitrile-water (60:40, by volume) as eluent at a flow rate of 16 mL / min to give chienhainanin I (8.0 mg).

[0046] (4) ①Structure identification of chienhainanin G

[0047] Colorless oil; UV (CH3OH) λ max (log ε) 227 (4.13), 266 (4.22); HR-ESI-MS showed quasi-molecular ion peak at m / z 315.2317 [M+H] + (calcd for C 21 H 31 O2 + : 315.2319). 1 H NMR and 13 C NMR data were listed in Table 1, and spectra were shown in Figs. Figures 1-2 .

[0048] According to the above physicochemical and spectral data, the structure of chienhainanin G was identified

[0049] Table 1. H NMR (600 MHz) and 1 C NMR (150 MHz) data of chienhainanin G 13

[0050]

[0051] ​Solvents are CDC13, δ units are ppm, J units are Hz

[0052] 2. Structure identification of Chienhainanin H

[0053] Colorless oil; UV(CH3OH) λ max (log ε) 227(4.12), 266(4.53), 276(4.42); HR-ESI-MS showed the quasi-molecular ion peak m / z 315.2317[M+H] + (calcd for C 21 H 31 O2 + : 315.2319); 1 H NMR and 13 C NMR data are shown in Table 2, and the spectra are shown in Figures 3-4 .

[0054] Table 2. Chienhainanin H 1 H NMR(400MHz) and 13 C NMR(100MHz) data

[0055]

[0056] Solvents are CD3OD, δ units are ppm, J units are Hz

[0057] 3. Structure identification of Chienhainanin I

[0058] Colorless oil; UV(CH3OH) λ max (log ε) 207(4.60), 214(4.65), 239(4.00), 253(4.13), 267(4.29), 283(4.18); HR-ESI-MS showed the quasi-molecular ion peak m / z 315.2317[M+H] + (calcd for C 21 H 31 O2 + : 315.2319); 1 H NMR and 13 C NMR data are shown in Table 3, and the spectra are shown in Figures 5-6 .

[0059] Table 3. Chienhainanin I 1 H NMR(400MHz) and 13 C NMR(100MHz) data

[0060]

[0061]

[0062] Solvent is CD3OD, δ unit is ppm, J unit is Hz

[0063] (4) Structure identification of Chienhainanin J

[0064] Colorless oil; UV (CH3OH) λ max (log ε) 208 (4.49), 213 (4.52), 239 (3.93), 252 (4.05), 267 (4.20), 283 (4.10); HR-ESI-MS shows quasi-molecular ion peak m / z 315.2315 [M+H] + (calcd for C 21 H 31 O2 + : 315.2319); 1 HNMR and 13 C NMR data are shown in Table 4, and the spectrum is shown in Figures 7-8 .

[0065] Table 4. Chienhainanin J 1 HNMR (400MHz) and 13 C NMR (100MHz) data

[0066]

[0067] Solvent is CD3OD, δ unit is ppm, J unit is Hz

[0068] (5) Structure identification of Chienhainanin K

[0069] Colorless oil; UV (CH3OH) λ max (log ε) 214 (4.58), 240 (4.01), 253 (4.14), 267 (4.30), 283 (4.19); HR-ESI-MS shows quasi-molecular ion peak m / z 313.2160 [M+H] + (calcd for C 21 H 29 O2 + : 313.2162); 1 HNMR and 13 C NMR data are shown in Table 5, and the spectrum is shown in Figures 9-10 .

[0070] Table 5. Chienhainanin K 1H NMR (400 MHz) and 13 C NMR (100 MHz) data

[0071]

[0072] Solvent: CD3OD, δ unit: ppm, J unit: Hz

[0073] Structure identification of Chienhainanin L

[0074] Colorless oil; UV (CH3OH) λ max (log ε) 208 (4.29), 214 (4.37), 240 (3.68), 253 (3.80), 267 (3.97), 283 (3.86); HR-ESI-MS showed quasi-molecular ion peak m / z 313.2159 [M+H] + (calcd for C 21 H 29 O2 + : 313.2162); 1 H NMR and 13 C NMR data are shown in Table 6, and the spectrum is shown in Figures 11-12 .

[0075] Table 6. Chienhainanin L 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data

[0076]

[0077]

[0078] Solvent: CD3OD, δ unit: ppm, J unit: Hz

[0079] Example 2 Inhibition of Chienhainanins G-L on various bacteria

[0080] Enterococcus faecalis (ATCC 29212), methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were purchased from Hefei Allwile Biological Technology Co., Ltd.

[0081] The antibacterial activity of some compounds was tested using the broth microdilution method, with vancomycin and ciprofloxacin as positive control drugs. The specific procedure is as follows:

[0082] The test compound and positive control were dissolved in DMSO to prepare stock solutions of 12800 μg / mL. These stock solutions were serially diluted with CAMHB medium to 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, and then added sequentially to 96-well U-shaped plates at 100 μL per well, with three replicates per concentration. The test bacterial strain cells were also prepared to a concentration of 2 × 10⁻⁶ cells / well using CAMHB medium. 5 For a bacterial culture concentration of 100 μL / mL, add 100 μL of the bacterial culture to each well of a U-shaped 96-well plate and mix with the test compound and positive control drug to achieve a final bacterial concentration of 1 × 10⁻⁶. 5 The concentrations of the test compound and positive control were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL. A blank medium without bacterial suspension and test compound was used as a blank control, and a medium containing bacterial suspension was used as a negative control. After incubating the 96-well plate at 35°C for 24 hours, the minimum inhibitory concentration (MIC) of the test compound was read by visual observation.

[0083] Table 2. Inhibitory effects of Chienhainanins GL on various bacteria

[0084]

[0085] The results showed that the compound Chienhainanins GL had significant antibacterial activity against methicillin-resistant Staphylococcus aureus and Enterococcus faecalis, with MIC values ​​of 2–16 μg / mL.

[0086] In particular, the above-mentioned compounds exhibit significant antibacterial activity against methicillin-resistant Staphylococcus aureus, which is of great significance for the development of these compounds as novel drugs against drug-resistant bacteria.

[0087] The Chienhainanins GL of the present invention is a novel chemical entity with a molecular structure completely different from existing antibacterial drugs. It has good activity against drug-resistant bacteria and has the potential to be developed into a new type of antibacterial drug.

[0088] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An alkyne compound represented by formulae 1-6 or a pharmaceutically acceptable salt thereof:

2. The method for preparing the alkyne compound according to claim 1, characterized in that, comprising the following steps: (1) Chieniodendron hainanense (Merr.) Tsiang & P.T.Li branches and leaves are crushed into coarse powder, and then extracted with ethanol by percolation. The percolation liquid is combined and concentrated under reduced pressure until there is no alcohol smell to obtain total extract; (2) The total extract is mixed with 100-200 mesh silica gel and then extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract, respectively; (3) The dichloromethane extract is subjected to silica gel column chromatography, and gradient elution is performed with petroleum ether-ethyl acetate 100:1-0:100 as eluent. Similar fractions are combined after thin layer chromatography analysis to obtain 24 main fractions Fr.1-Fr.24; (4) Fractions Fr.15 and Fr.16 of the petroleum ether / ethyl acetate 100:20 elution part are combined and then subjected to silica gel column chromatography, and gradient elution is performed with petroleum ether-ethyl acetate 15:1-1:1 as eluent to obtain 6 sub-fractions Fr.15A-Fr.15F; (5) Fraction Fr.15D of the petroleum ether / ethyl acetate 12:1-7:1 elution part is subjected to octadecyl-bonded silica gel column chromatography, and gradient elution is performed with methanol-water 40:60-100:0 as eluent to obtain 20 fractions Fr.15Da-Fr.15Dt; (6) Fraction Fr.15Do of the methanol-water 70:30 elution part is separated and purified by reverse phase preparative HPLC with acetonitrile-water in a volume ratio of 50-60:50-40 as eluent to obtain 7 fractions Fr.15Do1-Fr.15Do7; (7) Fraction Fr.15Dp of the methanol-water 75:25 elution part is separated and purified by reverse phase preparative HPLC with acetonitrile-water in a volume ratio of 50-60:50-40 as eluent to obtain 7 fractions Fr.15Dp1-Fr.15Dp7; (8) Fraction Fr.15Do1 is separated and purified by reverse phase preparative HPLC with methanol-water in a volume ratio of 75:25 as eluent to obtain compound 1 and compound 6; Fraction Fr.15Dp1 is separated and purified by reverse phase preparative HPLC with acetonitrile-water in a volume ratio of 55:45 as eluent to obtain compound 5; Fraction Fr.15Dp2 is separated and purified by reverse phase preparative HPLC with methanol-water in a volume ratio of 75:25 as eluent to obtain compound 2; Fraction Fr.15Dp3 is separated and purified by reverse phase preparative HPLC with acetonitrile-water in a volume ratio of 60:40 as eluent to obtain compound 4; Fraction Fr.15Dp4 is separated and purified by reverse phase preparative HPLC with acetonitrile-water in a volume ratio of 60:40 as eluent to obtain compound 3.

3. The production method according to claim 2, characterized by, In the step (1), the concentration of ethanol is 90-95%.

4. A pharmaceutical composition comprising the alkyne compound or a pharmaceutically acceptable salt thereof according to claim 1.

5. Use of the acetylenic compound or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of an antibacterial medicament.

6. Use of the pharmaceutical composition according to claim 4 for the manufacture of an antibacterial medicament.

7. Use according to claim 5 or 6, characterised in that, The bacteria is one of methicillin-resistant Staphylococcus aureus or Enterococcus faecalis.

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