Nucleoside antibiotic with mycobacterium tuberculosis resisting activity and preparation method and application thereof

Abstract

The invention discloses a nucleoside antibiotic with anti-mycobacterium tuberculosis activity and a preparation method and application thereof. The nucleoside antibiotic is muramycin D9 which is extracted and separated from fermentation liquor of streptomyces sp. NRRL 30475, the molecular formula of the nucleoside antibiotic is C38H61N11O15, and the nucleoside antibiotic is a muramycin compound which is obtained by dehydrating and cyclizing valine at the terminal of a peptide chain and non-protein source amino acid epicpremycine imine for the first time. An in-vitro anti-mycobacterium tuberculosis activity research shows that the nucleoside compound provided by the invention has very strong antibacterial activity on mycobacterium smegmatis and is expected to be developed into a novel anti-mycobacterium tuberculosis medicine.

Description

Technical Field

[0001] The invention relates to a nucleoside antibiotic with anti-tuberculosis bacillus activity, and also relates to a preparation method and application of the nucleoside antibiotic. Background Art

[0002] Twelve years after penicillin was introduced as the first antibiotic, drug-resistant bacteria gradually emerged and even developed into super-resistant bacteria. With the widespread use of antibiotics, bacterial resistance continues to threaten human health and global public health security. In 2002, McDonald et al. first isolated 22 muraymycins from the fermentation broth of Streptomyces sp. NRRL 30471 (J Am. Chem. Soc. 2002, 124: 10260), and found that they have extremely strong MraY translocase activity, which can effectively inhibit the bacterial cell wall biosynthesis process, thereby showing strong antibacterial activity. Muraymycins belong to the nucleoside antibiotics. Their structural units mainly include valine, L-leucine, non-protein amino acid epicapreomycidine, aminopropane, and glycyluridine (GlyU) core parts. According to the structural variation of the leucine residues in the polypeptide part, they can be divided into four categories: A, B, C, and D. Category D is the simplest L-leucine, category C is (3S)-3-OH-L-leucine, and category B is L-leucine acylated and connected to a saturated fatty acid branch chain with a carbon chain length of 3 to 10 Cs. Category A is similar to category B, except that the end of the fatty acid branch chain is substituted by a guanidine group. Among them, category A has the strongest antibacterial activity, with a MIC of less than 0.03μg / mL for Escherichia coli mutants (E.coliΔtolC) and an ED of less than 0.01μg / mL for mice infected with Staphylococcus aureus. 50 In addition to its MraY translocase inhibitory activity, muraymycin D1 also has inhibitory activity against N-acetylglucosamine-1-phosphotransferase (WecA) of Mycobacterium tuberculosis in both replicating and non-replicating states, showing good anti-Mycobacterium tuberculosis activity. However, the yield of muraymycins in fermentation culture is low, and it is necessary to optimize the fermentation method to increase the yield of the compound, or to find other analogues with novel structures and stronger activities. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a nucleoside antibiotic having anti-tuberculosis activity, and also to provide a preparation method and application of the above nucleoside antibiotic.

[0004] Technical solution: The present invention discloses a nucleoside antibiotic with anti-tuberculosis activity, the molecular formula of which is C 38 H 61 N11 O 15 , named muraymycin D9, with a structure as shown in Formula I:

[0005]

[0006] The nucleoside antibiotic is a muraymycin nucleoside antibiotic formed by dehydration and cyclization of valine at the end of the peptide chain and epicapreomycidine imine, a non-proteinogenic amino acid; the nucleoside antibiotic is obtained by screening and optimizing the fermentation medium of Streptomyces sp. NRRL 30475 and its mutants, and then fermenting in large quantities and separating and purifying; or by chemical synthesis.

[0007] The preparation method of the above nucleoside antibiotics comprises the following steps:

[0008] (1) Fermentation: Take Streptomyces sp. NRRL 30475 strain, culture it in BPM23A medium to obtain seed culture solution, inoculate the seed culture solution into BPM23A medium to obtain fermentation solution;

[0009] (2) Extraction: centrifuging the fermentation liquid obtained in step (1) to obtain mycelium and supernatant; extracting the mycelium with methanol by ultrasonic extraction, and obtaining extract A after concentration; adding non-ionic macroporous resin to the supernatant, stirring, filtering, discarding the filtrate, eluting the macroporous resin with methanol until it is colorless, and recovering methanol to obtain extract B;

[0010] (3) Separation: Extracts A and B are combined and separated and purified by reversed phase MCI column chromatography, Sephadex LH-20 column chromatography and preparative liquid chromatography to obtain nucleoside antibiotics with anti-tuberculosis activity.

[0011] Specifically, step (1) comprises taking a purified M2 plate of Streptomyces sp. NRRL 30475 strain and culturing it with BPM23A medium at 25 to 30° C. for 3 to 5 days; inoculating the obtained seed culture solution into 10 to 15 L of BPM23A medium, and culturing it at 25 to 30° C. for 7 to 10 days to obtain a fermentation solution.

[0012] Wherein, in step (2), the mycelium is extracted 3 to 5 times by ultrasonic extraction with methanol; 1% to 4% of non-ionic macroporous resin is added to the supernatant, and the mixture is stirred for 6 to 8 hours and then filtered.

[0013] Among them, step (3) is specifically as follows: the above-mentioned extract is separated by MCI column chromatography, and eluted with 20%, 40%, 60%, 80% and 100% methanol in sequence, and a total of 19 fractions Fr.1-1 to Fr.1-19 are obtained by combining; Fr.1-7 is separated by gel column chromatography to obtain 5 fractions Fr.1-7-1 to Fr.1-7-5, and Fr.1-7-2 is eluted by preparative liquid phase MeOH to obtain nucleoside antibiotics with anti-tuberculosis activity.

[0014] The invention also discloses a pharmaceutical composition, comprising the above nucleoside antibiotics having anti-tuberculosis bacillus activity and a pharmaceutically acceptable carrier.

[0015] The above nucleoside antibiotics having anti-tuberculosis activity can be used in the preparation of antibacterial drugs.

[0016] Wherein, the antibacterial drug is an anti-tuberculosis drug.

[0017] Wherein, the drug is in the form of tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, pills, ointments or transdermal controlled-release patches.

[0018] When the nucleoside compounds provided by the present invention are made into tablets, muraymycin D9 and lactose or corn starch are mixed evenly, and lubricant magnesium stearate is added when necessary, and then tablets are pressed to form tablets; when the nucleoside compounds provided by the present invention are made into capsules, muraymycin D9 and carrier lactose or corn starch are mixed evenly, and then granulated, and then capsules are made to form capsules; when the nucleoside compounds provided by the present invention are made into granules, muraymycin D9 and diluent lactose or corn starch are mixed evenly, granulated, and dried to form granules; when the nucleoside compounds provided by the present invention are made into powder injections and injections, a carrier is added to prepare them according to conventional pharmaceutical methods; when the nucleoside compounds provided by the present invention are made into dosage forms such as fat emulsions, ointments or transdermal controlled release patches, a carrier is added to prepare them according to conventional pharmaceutical methods.

[0019] Principle of the invention: The nucleoside antibiotic with anti-tuberculosis activity of the present invention is obtained by screening and optimizing the fermentation conditions of Streptomyces sp.NRRL 30475, adding biosynthetic precursors such as leucine for large-scale fermentation, and using modern chromatography to separate and purify, thereby obtaining a novel muraymycin compound, which is the first reported muraymycin nucleoside antibiotic with dehydration cyclization of valine at the end of the peptide chain and non-protein amino acid epicapreomycidine imine, named muraymycin D9. The structural skeleton of the compound is rare and not easy to obtain through chemical synthesis or modification. The activity test results show that the compound muraymycin D9 has strong antibacterial activity against Mycobacterium smegmatis.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The nucleoside antibiotics with anti-tuberculosis activity of the present invention have shown, through in vitro anti-tuberculosis activity studies, that the nucleoside compound muraymycin D9 provided by the present invention has strong antibacterial activity against Mycobacterium smegmatis, IC 50 The concentration of the compound is 32 μg / mL, which is an excellent new antibacterial compound and can be developed into a new antibacterial drug. The drug is in the form of tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, pills, ointments or transdermal controlled release patches. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the structure of the nucleoside antibiotic muraymycin D9 of the present invention;

[0022] Figure 2 is the HRESI-MS graph of the nucleoside antibiotic muraymycin D9 of the present invention;

[0023] Figure 3 The nucleoside antibiotic muraymycin D9 of the present invention 1 H NMR spectra;

[0024] Figure 4 The nucleoside antibiotic muraymycin D9 of the present invention 13 C NMR spectra;

[0025] Figure 5 is the HSQC diagram of the nucleoside antibiotic muraymycin D9 of the present invention;

[0026] Figure 6 is the HMBC diagram of the nucleoside antibiotic muraymycin D9 of the present invention;

[0027] Figure 7 It is the NOESY diagram of the nucleoside antibiotic muraymycin D9 of the present invention. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can be purchased through conventional channels, among which Streptomyces sp. NRRL 30475 was purchased from ARS Culture Collection (https: / / nrrl.ncaur.usda.gov / ).

[0029] Example 1

[0030] The preparation method of the nucleoside antibiotic muraymycin D9 of the present invention comprises the following steps:

[0031] (1) Fermentation: A purified strain of Streptomyces sp. NRRL 30475 was cultured at 28°C for 3 days using BPM23A medium (medium composition: 5.0 g / L glucose; 20.0 g / L HY yeast 441; 7.0 g / L calcium carbonate; 80.0 g / L Maltrin m180; 1.5 g / L L-methionine; 1.5 g / L L-leucine; 1.5 g / L L-arginine). The obtained seed culture solution was inoculated into 10 L of BPM23A medium and cultured at 28°C for 7 days to obtain a fermentation solution.

[0032] (2) Extraction: The fermentation liquid was centrifuged to obtain mycelium and supernatant. The mycelium was directly extracted with methanol ultrasonically for 3 times, and extract A was obtained after concentration; 1%-4% non-ionic macroporous resin was added to the supernatant, stirred for 6-8 hours, filtered, and the filtrate was discarded. The macroporous resin was eluted with methanol until colorless, and methanol was recovered to obtain extract B; the results were analyzed by HPLC-MS, and A and B were combined for subsequent separation;

[0033] (3) Separation: The extract was separated by MCI column chromatography, eluted with 20%, 40%, 60%, 80% and 100% methanol in sequence, and 19 fractions (Fr.1-1 to Fr.1-19) were obtained. Fr.1-7 was separated by gel column chromatography to obtain 5 fractions (Fr.1-7-1 to Fr.1-7-5), and Fr.1-7-2 was separated by preparative liquid chromatography (eluted with 26% MeOH) to obtain the compound muraymycin D9 (yield: 4.6 mg), the structural formula of which is as follows: Figure 1 shown.

[0034] The nucleoside compound muraymycin D9 of the present invention is a white powder, and the electrospray ionization mass spectrometry signal m / z912.4427[M+H] + and m / z 910.3[MH] - , its molecular formula is inferred to be C 38 H 61 N 11 O 15 . 1 H and 13 C NMR data are shown in Table 1.

[0035] Table 1. Muraymycin D9 1 H and 13 C NMR data (D2O)

[0036]

[0037]

[0038] Structural analysis of muraymycin D9: yellow powder, according to HRESIMS data (m / z: 912.4427 [M+H] + , the calculated value is 912.4427, Figure 2 ) It is speculated that its molecular formula is C 38 H 61 N 11 O 15 The degree of unsaturation is 14. 1 HNMR, 13 C NMR and HSQC spectra ( Figure 3 to Figure 5 ) were analyzed and found to contain 7 carbonyl groups, 1 guanidine group, 5 methyl groups, 7 methylene groups, 18 methine groups (including 2 sp 2 Compared with the known compound muraymycin D1, its molecular formula has one less H2O and one more unsaturation. C 157.1), C-33(δ C 175.7) shifted to high field; C-34 (δ C 62.9) shifted downfield and bound to H-27 of muraymycin D9 [δ H 4.82 (1H, m)] correlates with the key HMBC of C-33 ( Figure 6 ), and its planar structure was determined to be the first reported muraymycin compound in which the carboxyl group of the terminal valine was dehydrated and cyclized with the non-proteinogenic amino acid epicapreomycidine imine.

[0039] By comparing the NMR data (Table 1) and combining the NOESY related signals ( Figure 7 ) and the shared biosynthetic pathway, the relative configuration of the compound muraymycin D9 was determined as follows: Figure 1 The new compound shown.

[0040] Example 2

[0041] Evaluation of anti-Mycobacterium smegmatis activity:

[0042] Take the Mycobacterium smegmatis MC2155 strain, streak culture with Middlebrook 7H10 solid medium, place at 37°C, culture for 2 days, then inoculate into sterile 7H9 liquid medium, shake culture at 37°C for 24 hours until the strain reaches the logarithmic growth phase, and perform subsequent MIC determination experiments. The cultured mycobacteria were centrifuged at 5000rpm×10min to collect the precipitate, washed twice with 7H9 medium, resuspended in fresh 7H9 medium, and ultrasonically dispersed. The OD of Mycobacterium smegmatis was 0.04477 μg / mL. 600 Adjust to 0.02 for later use. In a transparent 96-well plate, dilute the drug to be screened in a 2-fold gradient, set up 4 replicate wells, and set up strain growth controls and culture medium controls. For Mycobacterium smegmatis, the positive control drug is isoniazid. The specific steps are as follows: Use a shotgun to take 200μl / well of fresh culture medium and add it to the first column, take 100μl / well of fresh culture medium and add it to columns 2 to 11, and use a shotgun to add 200μl to each of the four EFGH wells in column 12 as a culture medium control. In the wells in the first column, add 2μl of the 12.8mg / ml compound stock solution prepared with DMSO. Dilute to the 11th column by gradient dilution method, and then take out 100μl of liquid from the 11th column. At this point, the wells in columns 1 to 11 all contain 100μl of gradient diluted compound mixture. Subsequently, add 100μl OD 600 = 0.02 of Mycobacterium smegmatis, making the final concentration 0.01. Add 200 μl of bacterial suspension to each of the four wells ABCD in the 12th column as a bacterial liquid control. At this point, each well of the entire 96-well plate has 200 μl of liquid. Finally, place it directly in a 37°C incubator and culture for 2 days, and use an enzyme marker to measure the bacterial turbidity OD 600 .

[0043] The results showed that the IC of muraymycin D9 against Mycobacterium smegmatis 50The compound has inhibitory activity against Mycobacterium smegmatis in vitro at 32 μg / mL. Mycobacterium smegmatis is often used as a surrogate model organism in the study of Mycobacterium tuberculosis. Although it has a high degree of sequence homology with M. tuberculosis, it is considered non-pathogenic to humans and is therefore widely used to study Mycobacterium tuberculosis and other in vivo infections, and is often used to evaluate the potential therapeutic applications of drugs.

[0044] Example 3

[0045] Preparation of tablets:

[0046] Take the muraymycin D9 prepared in Example 1 above, add appropriate amounts of pharmaceutical excipients such as starch and magnesium stearate, mix well, and press into tablets to make tablets for oral use.

[0047] Example 4

[0048] Preparation of capsules:

[0049] Take the muraymycin D9 prepared in Example 1 above, add an appropriate amount of starch as a pharmaceutical excipient, mix thoroughly, and then put it into capsules to prepare capsules for oral use.

[0050] Example 5

[0051] Preparation of granules:

[0052] Take the muraymycin D9 prepared in Example 1 above and add an appropriate amount of corn starch as a pharmaceutical excipient, mix thoroughly, granulate, and dry to prepare granules for oral use.

[0053] Therefore, the nucleoside antibiotics with anti-tuberculosis activity of the present invention have strong antibacterial activity against Mycobacterium smegmatis and are expected to be developed into new anti-tuberculosis drugs.

Claims

1. A nucleoside antibiotic having anti-tuberculosis activity, characterized in that: The molecular formula of the nucleoside antibiotic is C 38 H 61 N 11 O 15 , the structure is shown in Formula I:

2. The nucleoside antibiotic according to claim 1, characterized in that The nucleoside antibiotic is a muraymycin nucleoside antibiotic obtained by dehydration cyclization of valine at the end of the peptide chain and epicapreomycidine imine, a non-proteinogenic amino acid.

3. A method for preparing the nucleoside antibiotics according to claim 1, characterized in that: The following steps are involved: (1) Fermentation: Take Streptomyces sp. NRRL 30475 strain, culture it in BPM23A medium to obtain seed culture solution, inoculate the seed culture solution into BPM23A medium to obtain fermentation solution; (2) Extraction: centrifuging the fermentation liquid obtained in step (1) to obtain mycelium and supernatant; extracting the mycelium with methanol by ultrasonic extraction, and obtaining extract A after concentration; adding non-ionic macroporous resin to the supernatant, stirring, filtering, discarding the filtrate, eluting the macroporous resin with methanol until it is colorless, and recovering methanol to obtain extract B; (3) Separation: Extracts A and B are combined and separated and purified by reversed phase MCI column chromatography, Sephadex LH-20 column chromatography and preparative liquid chromatography to obtain nucleoside antibiotics with anti-tuberculosis activity.

4. The preparation method according to claim 3, characterized in that: Step (1) specifically comprises taking a purified M2 plate of Streptomyces sp. NRRL 30475 strain, culturing it with BPM23A medium at 25-30° C. for 3-5 days; inoculating the obtained seed culture solution into 10-15 L of BPM23A medium, culturing it at 25-30° C. for 7-10 days, and obtaining a fermentation solution.

5. The preparation method according to claim 3, characterized in that: In step (2), the mycelium is extracted 3 to 5 times by ultrasonic extraction with methanol; 1% to 4% of non-ionic macroporous resin is added to the supernatant, and the mixture is stirred for 6 to 8 hours and then filtered.

6. The preparation method according to claim 3, characterized in that: Step (3) is specifically as follows: the extract is separated by MCI column chromatography, and eluted with 20%, 40%, 60%, 80% and 100% methanol in sequence, and a total of 19 fractions Fr.1-1 to Fr.1-19 are obtained; Fr.1-7 is separated by gel column chromatography to obtain 5 fractions Fr.1-7-1 to Fr.1-7-5, and Fr.1-7-2 is eluted by preparative liquid phase MeOH to obtain nucleoside antibiotics with anti-tuberculosis activity.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleoside antibiotic with anti-tuberculosis activity according to claim 1, and a pharmaceutically acceptable carrier.

8. Use of the nucleoside antibiotics having anti-tuberculosis activity according to claim 1 in the preparation of antibacterial drugs.

9. The use according to claim 8, characterized in that: The antibacterial drug is an anti-tuberculosis drug.

10. The use according to claim 8, characterized in that: The drug is in the form of tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, dropping pills, ointments or transdermal controlled-release patches.

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