Innovamycin structure derivatives with anti-tuberculosis activity

Abstract

The invention relates to a group of creatinamycin structure derivatives with anti-tuberculosis activity. The structure of the creatinamycin structure derivatives is shown in the specification. The invention further relates to application of the innovative mycin structure derivative with the anti-tuberculosis activity in preparation of drugs. The drugs are used for treating diseases caused by infection of drug-resistant mycobacterium tuberculosis, multi-drug-resistant mycobacterium tuberculosis and / or extensive drug-resistant mycobacterium tuberculosis. The invention further relates to application of the innovative mycin structure derivative with the anti-tuberculosis activity in preparation of drugs for treating diseases caused by infection of the drug-resistant mycobacterium tuberculosis, the multi-drug-resistant mycobacterium tuberculosis and / or the extensive drug-resistant mycobacterium tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a group of innovative mycin structural derivatives with anti-tuberculosis activity. Background Art

[0002] Tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis, continues to pose a significant global public health threat. According to the World Health Organization's "Global Tuberculosis Report 2024," TB incidence is showing a significant upward trend: 10.8 million new cases were confirmed globally in 2023, a continued increase from 10.6 million in 2022 and 10.3 million in 2021. Approximately 1.25 million TB-related deaths were expected worldwide in 2023, with a mortality rate nearly twice that of deaths related to HIV infection. The current standard treatment regimen, which utilizes quadruple therapy with rifampicin (RIF), isoniazid (INH), ethambutol, and pyrazinamide, is effective against drug-susceptible TB, but its effectiveness against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) Mycobacterium tuberculosis is significantly limited. Therefore, it is urgent to develop new anti-drug-resistant tuberculosis drugs.

[0003] Chuangxinmycin (CM), an indole alkaloid antibiotic with unique pharmacological properties, was discovered in the 1970s and was originally isolated from Actinoplanes tsinanensis CPCC200056. This compound, which has a dihydrothiopyrano[4,3,2-cd]indole structural framework, exhibits in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria. Its efficacy has been initially demonstrated in mouse infection models and in clinical trials against Escherichia coli and Shigella dysenteriae. CM exerts its antibacterial activity by selectively inhibiting tryptophanyl-tRNA synthetase (TrpRS), a target currently underexploited in clinical antimicrobial therapy. Recent studies have shown that CM exhibits excellent antibacterial activity against both the standard strain of Mycobacterium tuberculosis H37Rv and clinically resistant isolates (MIC = 0.78-1 μg / mL). Its structural analog, 3-demethylinnovamycin (DCM), also exhibited similar inhibitory activity against Mycobacterium tuberculosis (MIC = 0.78-4 μg / mL). However, it is noteworthy that the antibacterial activity of DCM and 3-methylinnovamycin (MCM) against most Gram-negative and Gram-positive bacteria (such as Escherichia coli and Staphylococcus aureus) was significantly reduced or even eliminated, indicating that the 3-methyl substituent plays a significant role in the broad-spectrum antibacterial activity of CM. DCM retained similar inhibitory activity against M. tuberculosis, suggesting a novel antibacterial mechanism. Therefore, innovamycin compounds are expected to become lead compounds for the development of new anti-tuberculosis drugs.

[0004] To enrich the diversity of novel structural derivatives of innovamycin and thereby enhance its potential for drug discovery and development, it is imperative to develop green and efficient synthetic methods for innovamycin derivatives. Compared to the numerous problems associated with traditional chemical synthesis of innovamycin and its derivatives, biosynthetic methods not only eliminate cumbersome synthetic routes, but also offer a green, efficient, and environmentally compatible synthesis process and can yield innovamycin with specific stereochemical configurations exhibiting antimicrobial activity. In recent years, the key biosynthetic mechanisms of innovamycin have been elucidated: starting with L-tryptophan, innovamycin is synthesized through transamination via aminotransferases, sulfur incorporation via sulfur carrier proteins and sulfurtransferases, reductase-mediated thioketone reduction, cytochrome P450-catalyzed cyclization, and methylation via methyltransferases. This elucidation of the biosynthetic mechanism of innovamycin provides a solid theoretical foundation for the biosynthesis of novel structural derivatives of innovamycin.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The present invention first relates to a group of innovative mycin structural derivatives with anti-tuberculosis activity, the structures of which are shown in the following table.

[0007]

[0008]

[0009] The present invention also relates to a method for preparing the innovative mycin structural derivative, comprising the following steps:

[0010] (1) Using A. tsinanensis CPCC 200056 as the production strain, its fresh spore suspension was evenly spread on a culture dish containing solid fermentation medium (containing 0.2 mM halogenated L-tryptophan) and incubated at 28°C;

[0011] (2) After the culture was collected, continuous extraction was performed with ethyl acetate, and the organic phases were combined and vacuum dried to obtain a crude extract;

[0012] (3) performing preliminary purification by normal phase silica gel column chromatography, using a mobile phase consisting of solvent A (0.1% acetic acid / dichloromethane) and solvent B (0.1% acetic acid / methanol), using a linear gradient elution, collecting the active fraction containing the innovative mycin structural derivative, and concentrating under reduced pressure to obtain a primary purified crude product;

[0013] (4) The crude product of the primary purification is subjected to secondary purification using a reverse phase C18 chromatography system, the mobile phase system is adjusted to solvent A (0.1% acetic acid / water) and solvent B (0.1% acetic acid / methanol), and linear gradient elution is used to collect the active fraction containing the innovative mycin structural derivative to obtain a secondary purified crude product;

[0014] (5) The secondary purified crude product is refined by a semi-preparative HPLC system to obtain the target product, which is the innovative mycin structural derivative.

[0015] Preferably, the halogenated L-tryptophan is 5-F-Trp, 6-F-Trp, 7-F-Trp, 6-Cl-Trp or 7-Cl-Trp; most preferably, it is 5-F-Trp.

[0016] Preferably,

[0017] In step (1), the solid-state fermentation medium is ISP2 medium, and the culture is statically cultured for 7 days;

[0018] In step (2), two consecutive extractions are performed using ethyl acetate with a volume twice that of the culture;

[0019] In step (3), a linear gradient of 0-10% B was used for elution;

[0020] In step (4), the gradient elution condition was set to 10-100% B;

[0021] In step (5), an Xselect CSH C18 OBD column (10×250 mm, 5 μm) was used for isocratic elution in acetonitrile-water (25:75, the aqueous phase containing 0.1% acetic acid).

[0022] The present invention also relates to the use of the innovative mycin structural derivative with anti-tuberculosis activity in the preparation of a medicament, wherein the medicament is used to treat diseases caused by Mycobacterium tuberculosis infection. Preferably, the Mycobacterium tuberculosis is rifampicin (RIF), isoniazid (INH) and / or linezolid (LZD)-resistant Mycobacterium tuberculosis, multidrug-resistant Mycobacterium tuberculosis and / or extensively drug-resistant Mycobacterium tuberculosis.

[0023] The present invention also relates to a pharmaceutical composition containing a therapeutically effective amount of the innovative mycin structural derivative with anti-tuberculosis activity, and the pharmaceutical composition also contains necessary pharmaceutical excipients.

[0024] The beneficial effects of the present invention are:

[0025] Based on the analysis of the biosynthetic mechanism of innovamycin, this study successfully obtained 11 halogenated innovamycin derivatives using a precursor-directed biosynthesis strategy. After separation and purification, six derivative monomer compounds were obtained. Their structures were confirmed by high-resolution mass spectrometry (HR-MS), high-resolution tandem mass spectrometry (HR-MS / MS), and nuclear magnetic resonance (NMR) spectroscopy. Antimicrobial activity was tested against a standard strain of Mycobacterium tuberculosis, H37Rv, and clinically isolated isoniazid- and rifampicin-resistant strains. Results showed that 5-F-CM, 7-F-DCM, and 6-Cl-CM / DCM exhibited significant inhibitory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 (A) LC-MS analysis of metabolites of CM-producing bacteria fed 5 / 6 / 7-F / Cl / Br-Trp; (B) Analysis of tryptophan derivative incorporation. Dark blue: Efficient incorporation, and the corresponding derivatives were isolated and purified; medium blue: Low-level incorporation, but the corresponding derivatives were not isolated and purified; light blue: No corresponding derivatives were detected.

[0027] Figure 2 、 5 (-)-HR-ESIMS mass spectrum of -F-CM

[0028] Figure 3 、 5(-)-HR-ESIMS / MS mass spectrum of -F-CM

[0029] Figure 4 、 5 -F-CM 1 H NMR spectrum (deuterated acetone, 600 MHz)

[0030] Figure 5 、 5 -F-CM 13 C NMR spectrum (deuterated acetone, 150 MHz)

[0031] Figure 6 、 5 HSQC spectrum of -F-CM (deuterated acetone)

[0032] Figure 7 、 5 -HMBC spectrum of F-CM (deuterated acetone)

[0033] Figure 8 、 5 -F-CM 1 H- 1 H COSY spectrum (deuterated acetone)

[0034] Figure 9 、 5 (-)-HR-ESIMS mass spectrum of -F-DCM

[0035] Figure 10 、 5 (-)-HR-ESIMS / MS mass spectrum of -F-DCM

[0036] Figure 11 、 5 -F-DCM 1 H NMR spectrum (deuterated methanol, 600 MHz)

[0037] Figure 12 、 5 -F-DCM 13 C NMR spectrum (deuterated methanol, 150 MHz)

[0038] Figure 13 、 5 HSQC spectrum of -F-DCM (deuterated methanol)

[0039] Figure 14 、 5 -HMBC spectrum of F-DCM (deuterated methanol)

[0040] Figure 15 、 5 -F-DCM1 H- 1 H COSY spectrum (deuterated methanol)

[0041] Figure 16 、 7 -F-DCM 1 H NMR spectrum (deuterated methanol, 600 MHz)

[0042] Figure 17 、 7 -F-DCM 13 C NMR spectrum (deuterated methanol, 150 MHz)

[0043] Figure 18 、 7 HSQC spectrum of -F-DCM (deuterated methanol)

[0044] Figure 19 、 7 -HMBC spectrum of F-DCM (deuterated methanol)

[0045] Figure 20 、 7 -F-DCM 1 H- 1 H COSY spectrum (deuterated methanol)

[0046] Figure 21 、 6 (-)-HR-ESIMS mass spectrum of -Cl-CM

[0047] Figure 22 、 6 (-)-HR-ESIMS / MS mass spectrum of -Cl-CM

[0048] Figure 23 、 6 (-)-HR-ESIMS mass spectrum of -Cl-DCM

[0049] Figure 24 、 6 (-)-HR-ESIMS / MS mass spectrum of -Cl-DCM

[0050] Figure 25 、 6 -Cl-DCM 1 H NMR spectrum (deuterated methanol, 600 MHz)

[0051] Figure 26 、 6 -Cl-DCM 13 C NMR spectrum (deuterated methanol, 150 MHz)

[0052] Figure 27 、 6 HSQC spectrum of -Cl-DCM (deuterated methanol)

[0053] Figure 28 、 6 HMBC spectrum of -Cl-DCM (deuterated methanol)

[0054] Figure 29 、 6 -Cl-DCM 1 H- 1 H COSY spectrum (deuterated methanol)

[0055] Figure 30 、 7 (-)-HR-ESIMS mass spectrum of -Cl-DCM

[0056] Figure 31 、 7 (-)-HR-ESIMS / MS mass spectrum of -Cl-DCM

[0057] Figure 32 、 7 -Cl-DCM 1 H NMR spectrum (deuterated methanol, 600 MHz)

[0058] Figure 33 、 7 -Cl-DCM 13 C NMR spectrum (deuterated methanol, 150 MHz)

[0059] Figure 34 、 7 HSQC spectrum of -Cl-DCM (deuterated methanol)

[0060] Figure 35 、 7 HMBC spectrum of -Cl-DCM (deuterated methanol)

[0061] Figure 36 、 7 -Cl-DCM 1 H- 1 H COSY spectrum (deuterated methanol) DETAILED DESCRIPTION

[0062] Experimental Materials

[0063] CM-producing bacteria: A. tsinanensis CPCC 200056, deposited in the China Pharmaceutical Microbiology Culture Collection Center.

[0064] Chuangxinmycin (CM) is a fermentation product of A. tsinanensis CPCC 200056 and was prepared in our laboratory.

[0065] 3-Demethylcinoma (DCM) is a fermentation product of A. tsinanensis CPCC 200056 and was prepared in our laboratory.

[0066] M. phlei was obtained from China Pharmaceutical Microorganism Culture Collection Center, CPCC 160023.

[0067] M. smegmatis was obtained from China Pharmaceutical Microorganism Culture Collection Center, CPCC 160024.

[0068] M.tuberculosis H37Rv, M.tuberculosis M9483, M.tuberculosis M6600, and M.tuberculosis M3551 were preserved and subjected to relevant activity assays by Professor Zhang Tianyu's laboratory at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences.

[0069] B. subtilis, S. aureus, E. coliΔtolC, and P. aeruginosa 11 were maintained by our laboratory.

[0070] Example 1: Precursor-directed biosynthesis of halogenated innovative mycin

[0071] The general synthesis method in this embodiment is briefly described as follows:

[0072] Halogenation plays an important role in the optimization of drug molecules, such as regulating the lipid-water partition coefficient, improving metabolic stability, and optimizing pharmacokinetic properties. Based on the fact that the biosynthesis of innovative mycin starts with L-Trp (L-tryptophan), we first attempted to feed 6-Cl-Trp (L-tryptophan substituted with Cl at the 6th position, the same below) to the CM-producing bacterium A. tsinanensis CPCC 200056 and then used high-performance liquid chromatography-mass spectrometry (LC-MS) to detect the production of new compounds. The specific method is as follows:

[0073] (1) A. tsinanensis CPCC 200056 was inoculated into ISP2 medium (6 cm in diameter) containing 0.2 mM halogenated L-tryptophan (6-Cl-Trp) and fermented at 28°C for 7 days.

[0074] (2) After fermentation, the culture was cut into 1 × 1 cm cubes and extracted with two volumes of ethyl acetate for 48 h. The organic phase was evaporated and redissolved in methanol and filtered through a 0.22 μm organic filter membrane to prepare the test sample.

[0075] (3) An Agilent 1100-6410 triple quadrupole liquid chromatography-mass spectrometry (HPLC-ESI-MS) system was used for analysis. Chromatographic separation was performed on a ZORBAX SB-C8 column (4.6×250 mm, 5 μm) with a flow rate of 0.8 mL / min, a detection wavelength of 230 nm, and a column temperature of 25°C. An acetonitrile-water (containing 0.1% glacial acetic acid) gradient elution program (0-30 min, acetonitrile ratio 15%-100%) was used.

[0076] Two new compounds 6-Cl-DCM and 6-Cl-CM ( Figure 1 )

[0077] In order to obtain more derivatives, we fed a variety of tryptophan derivatives to the CM-producing bacteria, including L-tryptophan halogenated derivatives with different halogen atoms at different positions (such as 5 / 6 / 7-F / Cl / Br-Trp) and L-tryptophan derivatives with other substituents at different positions (such as 1 / 5 / 6 / 7-CH3 / OCH3 / CN / OH / BnO-Trp). LC-MS analysis showed that 5 / 6 / 7-F-Trp and 6 / 7-Cl-Trp were most easily incorporated into the innovative biosynthetic pathway to generate the corresponding halogenated derivatives ( Figure 1 However, as the substituent size increases, incorporation becomes increasingly difficult, especially for the 5-halogenated tryptophan, where neither 5-Cl-Trp nor 5-Br-Trp produces the corresponding derivatives. 6 / 7-Br-Trp can be incorporated and produce a small amount of the corresponding DCM derivative.

[0078] In addition, we found that the yield of DCM halogenated derivatives was higher than that of CM halogenated derivatives, which may be due to the substrate specificity of methyltransferase. Among the other tryptophan derivatives fed, such as 5-hydroxy / cyano / benzyloxy, 6-methoxy, 7-methyl and 1-methyl tryptophan, they failed to be incorporated into the biosynthetic pathway to generate the corresponding derivatives ( Figure 1 In general, the yield of fluorinated CM / DCM derivatives is higher than that of chlorinated derivatives. This study obtained 11 halogenated CM / DCM derivatives ( Figure 1 ).

[0079] Example 2: Isolation, purification, structural identification and activity confirmation of halogenated innovamycin derivatives

[0080] 1. Compound separation, purification and structure confirmation

[0081] To determine the structure of the halogenated derivative and evaluate its activity, we scaled up the fermentation to obtain sufficient quantities of the compound. The fermentation broth containing the target compound was first extracted with ethyl acetate (EtOAc) and concentrated by rotary evaporation to obtain a crude extract. The extract was then separated by normal-phase chromatography on a silica gel column and purified by semi-preparative high-performance liquid chromatography.

[0082] The specific method is as follows:

[0083] (1) Using A. tsinanensis CPCC 200056 as the production strain, its fresh spore suspension was evenly spread on a 15 cm culture dish containing 50 mL of fermentation medium (containing 0.2 mM 5-F-Trp) and cultured at 28°C for 7 days.

[0084] (2) After collecting about 5 L of culture, two consecutive extractions were performed using twice the volume of ethyl acetate (48 h each time), and the organic phases were combined and vacuum dried to obtain a crude extract.

[0085] (3) Preliminary purification was performed by normal phase silica gel column chromatography system, with the mobile phase consisting of solvent A (0.1% acetic acid / dichloromethane) and solvent B (0.1% acetic acid / methanol), using a 0-10% B linear gradient elution, collecting the active fractions containing innovomycin and its derivatives, and concentrating under reduced pressure to obtain a crude product.

[0086] (4) Secondary purification was performed using a reversed-phase C18 chromatography system. The mobile phase system was adjusted to solvent A (0.1% acetic acid / water) and solvent B (0.1% acetic acid / methanol), and the gradient elution condition was set to 10-100% B to obtain a mixed component containing 5-F-CM and 5-F-DCM.

[0087] (5) The mixed components were purified by semi-preparative HPLC using an Xselect CSH C18 OBD column (10×250 mm, 5 μm) under isocratic elution conditions of acetonitrile-water (25:75, the aqueous phase containing 0.1% acetic acid). High-purity 5-F-CM (2.6 mg) and 5-F-DCM (0.9 mg) were finally separated.

[0088] By using the precursor feeding strategy, 0.2 mM 6-F-Trp, 7-F-Trp, 6-Cl-Trp or 7-Cl-Trp was replaced in step (1), and the fermentation and separation and purification processes were similar to those described above, wherein the isocratic elution ratio of the semi-preparative HPLC was adjusted accordingly according to the actual situation. Finally, 7-F-DCM (2.7 mg), 6-Cl-CM (0.33 mg), 6-Cl-DCM (1.53 mg) and 7-Cl-DCM (1.93 mg) were successfully obtained.

[0089] Six new innovative halogenated derivatives of oximes were identified, namely 5-F-CM, 5-F-DCM, 7-F-DCM, 6-Cl-CM, 6-Cl-DCM, and 7-F-DCM. Their structures were confirmed by electrospray ionization high-resolution mass spectrometry (ESI-HRMS), ESI-HRMS / MS, and nuclear magnetic resonance (NMR) spectroscopy (Table 1-2, Figure 2-Figure 36 ), but the amount of 6-Cl-CM obtained by separation and purification was insufficient and could not be further confirmed by NMR.

[0090] Table 1. NMR data of 5-F-CM, 5-F-DCM and 7-F-DCM

[0091]

[0092] Note: 5-F-CM (deuterated acetone, 600 MHz); 5-F-DCM and 7-F-DCM (deuterated methanol, 600 MHz)

[0093] Table 2. NMR data of 6-Cl-DCM and 7-Cl-DCM

[0094]

[0095] Note: 6-Cl-DCM and 7-Cl-DCM (deuterated methanol, 600 MHz)

[0096] 2. Evaluation of compound activity (the structures of the tested compounds are shown in Table 5)

[0097] For the determination of inhibitory activity against M. tuberculosis H37Rv and clinical isolates, the Alamar Blue microplate assay was used:

[0098] (1) The strain in the logarithmic growth phase was adjusted to 1×10 6 CFU / mL, 100 μL of bacterial suspension was added to a 96-well plate, and the experimental group was supplemented with 4 μL of gradient dilution solution (DMSO dissolved, final concentration gradient was 64-0.125 μg / mL).

[0099] (2) After 7 days of incubation at 37°C, a colorimetric reagent was added. The lowest drug concentration that completely inhibited the color change of Alamar Blue (blue to pink) was defined as the minimum inhibitory concentration (MIC). Three biological replicates were performed for each concentration. Isoniazid (INH), rifampicin (RIF), and linezolid (LZD) were used as positive controls.

[0100] For the evaluation of antimicrobial activity against other test strains, refer to the Clinical and Laboratory Standards Institute (CLSI) guidelines. [1-2]Establish a microdilution detection system. Inoculate the strain to be tested into Mueller-Hinton broth (MHB) [3] Prepare 1×10 6 CFU / mL bacterial suspension. After serial dilution of the test compound with MHB, 100 μL of the dilution was co-cultured with an equal volume of bacterial suspension in a 96-well plate. After incubation at 37°C for 10 hours, the minimum drug concentration (MIC) that inhibited bacterial growth was determined by visual observation. Three technical replicates were set for each concentration.

[0101] These include Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium phlei (M. phlei), Mycobacterium smegmatis (M. smegmatis) and some other strains.

[0102] The results are shown in Table 3 and Table 4.

[0103] (1) The minimum inhibitory concentration (MIC) values showed that 5-F-CM and 7-F-DCM had the strongest inhibitory activity against M. tuberculosis H37Rv, which was comparable to the activity of CM / DCM, while 5-F-DCM and 7-Cl-DCM completely lost their activity.

[0104] (2) CM and 5-F-CM also showed strong inhibitory activity against clinically isolated isoniazid / rifampicin-resistant Mycobacterium tuberculosis (information on strains M9483, M6600, and M3551 can be found in: Lu Y, Li Y, Fan J, Li X, Sun H, Wang L, Han X, Zhu Y, Zhang T, Shi Y, et al: Expanding structural diversity of 5'-aminouridine moiety of sansanmycin via mutational biosynthesis. Front Bioeng Biotechnol 2023, 11: 1278601.).

[0105] (3) In addition to showing certain antibacterial activity against Mycobacterium tuberculosis, 5-F-CM and 7-F-DCM had almost no inhibitory activity against other mycobacteria and common pathogens, suggesting that 5-F-CM has specific anti-tuberculosis activity.

[0106] Table 3 Anti-Mycobacterium tuberculosis and anti-other mycobacterial activities of halogenated innovative mycobacterium derivatives

[0107]

[0108]

[0109] Note: M. tuberculosis H37Rv is the standard strain; M6600, M3551 and M9483 are clinically isolated multidrug-resistant Mycobacterium tuberculosis strains, resistant to isoniazid (INH) and rifampicin (RIF), and sensitive to linezolid (LZD); " / " in the table indicates not tested; "R" indicates drug-resistant; "S" indicates sensitive; M. phlei is Mycobacterium smegmatis; M. smegmatis is Mycobacterium phlei.

[0110] Table 4 Antibacterial activity of halogenated innovative mycin derivatives against other common pathogenic microorganisms

[0111]

[0112] Bacillus subtilis: B.subtilis; Staphylococcus aureus: S.aureus; Escherichia coli: E.coliΔtolC; Pseudomonas aeruginosa: P.aeruginosa 11;

[0113] Table 5. Structural diagrams of various halogenated innovative mycin derivatives

[0114]

[0115]

[0116] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

[0117] References

[0118] [1] CLSI: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Eighth Edition. CLSI document M07-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

[0119] [2]Lu Y,Li Y,Fan J,Li X,Sun H,Wang L,et al.Expanding structuraldiversity of 5’-aminouridine moiety of sansanmycin via mutationalbiosynthesis.Front Bioeng Biotechnol.2023 Oct 30;11:1278601.

[0120] [3]Weissauer-Condon C,Engels I,Daschner FD.In vitro activity of fournew quinolones in Mueller-Hinton broth and peritoneal dialysis fluid.Eur JClin Microbiol.1987 Jun;6(3):324–6.

Claims

1. A group of innovative mycin derivatives with anti-tuberculosis activity, the structures of which are shown in the table below.

2. The method for preparing the innovative mycin structural derivative according to claim 1, comprising the following steps: (1) Using A. tsinanensis CPCC 200056 as the production strain, its fresh spore suspension was evenly spread on a culture dish containing solid fermentation medium (containing 0.2 mM halogenated L-tryptophan) and incubated at 28°C; (2) After the culture was collected, continuous extraction was performed with ethyl acetate, and the organic phases were combined and vacuum dried to obtain a crude extract; (3) performing preliminary purification by normal phase silica gel column chromatography, using a mobile phase consisting of solvent A (0.1% acetic acid / dichloromethane) and solvent B (0.1% acetic acid / methanol), using a linear gradient elution, collecting the active fraction containing the innovative mycin structural derivative, and concentrating under reduced pressure to obtain a primary purified crude product; (4) The crude product of the primary purification is subjected to secondary purification using a reverse phase C18 chromatography system, the mobile phase system is adjusted to solvent A (0.1% acetic acid / water) and solvent B (0.1% acetic acid / methanol), and linear gradient elution is used to collect the active fraction containing the innovative mycin structural derivative to obtain a secondary purified crude product; (5) The secondary purified crude product is refined by a semi-preparative HPLC system to obtain the target product, which is the innovative mycin structural derivative.

3. The method according to claim 2, characterized in that The halogenated L-tryptophan is 5-F-Trp, 6-F-Trp, 7-F-Trp, 6-Cl-Trp or 7-Cl-Trp, preferably 5-F-Trp.

4. The method according to claim 2 or 3, characterized in that In step (1), the solid-state fermentation medium is ISP2 medium, and the culture is statically cultured for 7 days; In step (2), two consecutive extractions are performed using ethyl acetate with a volume twice that of the culture; In step (3), a linear gradient elution of 0-10% B was used; In step (4), the gradient elution condition was set to 10-100% B; In step (5), an Xselect CSH C18 OBD column (10×250 mm, 5 μm) was used for isocratic elution in acetonitrile-water (25:75, the aqueous phase containing 0.1% acetic acid).

5. Use of the anti-tuberculosis active innovative mycin structural derivative according to claim 1 in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis infection, preferably, the Mycobacterium tuberculosis is rifampicin (RIF), isoniazid (INH) and / or linezolid (LZD)-resistant Mycobacterium tuberculosis, multidrug-resistant Mycobacterium tuberculosis and / or extensively drug-resistant Mycobacterium tuberculosis.

6. A pharmaceutical composition comprising a therapeutically effective amount of the innovative anti-tuberculosis derivative of claim 1, further comprising necessary pharmaceutical excipients.

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