CN111303247B

CN111303247B - Marine cyclopeptide compound and application thereof in preparation of mycobacterium tuberculosis resistant medicines

Info

Publication number: CN111303247B
Application number: CN201911149125.XA
Authority: CN
Inventors: 鞠建华; 张天宇; 孙长利; 刘志永; 马俊英
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2022-03-22
Anticipated expiration: 2039-11-21
Also published as: CN111303247A

Abstract

The invention discloses a marine cyclic peptide compound and its application in the preparation of anti-tuberculosis mycobacterial drugs. The new cyclic peptide compound Ilamycins G-R is shown in formula I. The present invention separates and obtains 12 cyclic peptide compounds from the fermentation culture of Streptomyces atratus SCSIO Zh16ΔilaR, all of which have significant inhibitory effects on Mycobacterium tuberculosis, and can be used for preparing anti-tuberculosis drugs and treating tuberculosis. The invention provides candidate compounds for the development of new anti-tuberculosis drugs, and has important significance for the development of marine drug resources in China.

Description

Marine cyclopeptide compound and application thereof in preparation of mycobacterium tuberculosis resistant medicines

Technical Field

The invention belongs to the field of natural products, and particularly relates to a marine streptomyces-derived cyclopeptide compound and application thereof in preparation of anti-mycobacterium tuberculosis drugs.

Background

Cyclic peptides are widely distributed in microorganisms, higher plants, and marine organisms (curr. protein. pept. sci.2004,5: 297. 315; mar. drug.2010,8: 810. 834; mar. drug.2012, 10: 963. 986; curr. opin. chem. biol.2017,38: 24-29). Due to a plurality of structural characteristics such as large surface area and rigid conformation, the cyclic peptide compound has various characteristics such as better target selectivity, affinity and metabolic stability, becomes an important source of the drug, and plays an important role in various drug treatments such as anti-tumor, anti-infection, anti-malaria and immunosuppression (curr. org. chem.2019,23: 38-75; curr. Opin. chem. Bio.2017,38: 24-29; drug. Discov. Today.2015,20: 122-. Wherein the cyclic peptide derived from a microorganism is synthesized by a non-ribosomal peptide synthetase. The unique modular composition of non-ribosomal peptide synthetases, as well as various pre-and post-modification enzymes, directly contribute to the complex structure and diverse activities of cyclic peptide compounds (chem. Rev.2006,106: 3468-3496; chem. biol.1999,6: R39-R48).

Riomycin (Ilamycins), also known as rifamycins (rufomycins), is a class of cyclic heptapeptide compounds originally isolated from Streptomyces atratus and Streptomyces islandicus (Nat. Commun.2017,8: 391; J. Antibiot. SerB.1963,16: 93-98; J. Antibiot.1963,16: 175-. The structure of the compound contains three unnatural amino acids, including N-substituted tryptophan, 2-amino-4-hexenoic acid and 3-nitrotyrosine. Wherein, the bleomycin C and the bleomycin E can cause triple negative breast cancer cell apoptosis through different pathways (J.Hematol.Oncol.2019,12: 60; int.J.biol.Sci.2019,15: 1723-1732). The bleomycin class of compounds can also exert their anti-tubercular activity by inhibiting ClpC1 and subsequently modulating intracellular protein degradation (antimicrob. Agents. Ch.2019,63: e2204-e 2218). In addition, many documents have been reported about the biosynthetic gene cluster of the bleomycin-type compound and the biosynthesis process thereof, as well as pre-modification and post-modification of the compound (nat. Commun.2017,8: 391).

The invention content is as follows:

one of the objects of the present invention is to provide a novel cyclic peptide compound of formula I, Ilamycins G-R (1-12):

the invention also aims to provide application of a marine actinomycete mutant strain (Streptomyces atratus SCSIO Zh16 delta ilaR) in preparation of Ilamycins G-R (1-12).

It is a further object of the present invention to provide a process for producing Ilamycins G-R (1-12) from a fermentation culture of Streptomyces atratus SCSIO Zh16 Δ ilaR, a marine actinomycete mutant.

According to the invention, preferably the Ilamycins G-R (1-12) is prepared from a fermentation culture of a marine actinomycete mutant strain (Streptomyces atratus SCSIO Zh16 Δ ilaR), and specifically comprises the following steps:

a. preparing a fermentation culture of Streptomyces atratus SCSIO ZH16 delta ilaR as a marine actinomycete mutant strain;

b. adding macroporous resin into the fermentation culture, stirring and adsorbing the compound in the fermentation broth, and filtering to obtain a mixture of the macroporous resin and the mycelium; soaking the mixture in EtOH for extraction, concentrating the extractive solution under reduced pressure to obtain extract, subjecting the extract to silica gel column chromatography, and purifying with CHCl₃Gradient elution is carried out on/MeOH 100/0,98/2,96/4,94/6,92/8,90/10,80/20 and 50/50v/v, a fraction Afr.3 eluted by chloroform-methanol with a volume ratio of 94:6 is collected, and the AFr.3 is separated and purified to obtain the compounds 1-12.

The fermentation culture for preparing the marine actinomycete mutant strain (Streptomyces atratus SCSIO ZH16 Delta ilaR) is prepared by the following method:

inoculating a marine actinomycete mutant strain (Streptomyces atraus SCSIO ZH 16. delta. ilaR) into a seed culture medium, fermenting to obtain a seed culture solution, inoculating the seed culture solution into a fermentation culture medium, and fermenting to obtain a fermentation culture. The seed culture medium contains per liter: 15g of soluble starch, 15g of soybean peptone FP410,5g of soybean meal, 15g of glycerol, 2g of CaCO3 and 30g of sea salt, and adjusting the pH value to 7.2-7.4.

The fourth purpose of the invention is to provide the application of the compound Ilamycins G-R (1-12), or the medicinal salt thereof in preparing antimycobacterial medicines.

An antimycobacterial agent comprising an effective amount of the aforementioned Ilamycins G-R (1-12), or a pharmaceutically acceptable salt thereof as an active ingredient.

Preferably, the antimycobacterial agent is an antituberculous mycobacterium agent.

The invention separates 12 cyclic peptide compounds from the fermentation culture of actinomyces marinus mutant strain Streptomyces atratus SCSIO ZH16 delta ilaR, all have obvious inhibiting effect on mycobacterium tuberculosis, can be used for preparing antituberculosis drugs and treating tuberculosis, thus the invention provides an alternative compound for developing new antituberculosis drugs and has important significance for developing Chinese marine drug resources.

The marine actinomycete mutant strain Streptomyces atratus SCSIO Zh16 delta ilaR is a mutant strain of marine Streptomyces atratus SCSIO Zh16 with ilaR gene deletion, and the specific construction method is shown in the patent number: 201610885104.4, title of the invention: the invention relates to a genetic engineering strain for directionally producing antitubercular active and antitumor active compounds and application thereof, wherein a marine actinomyces mutant strain Streptomyces atratus SCSIO Zh16 delta ilaR is the double-exchange mutant strain delta ilaR in the patent.

The starting strain Streptomyces atrophaeus SCSIO ZH16 of the present invention has been stored in the common microorganism center of the china committee for culture collection of microorganisms (CGMCC) 3, 10 days 2016, with the address: the postcode 100101 of institute for microbiology, china academy of sciences, west road No.1, north chen, chaoyang, beijing, the strain preservation number is: CGMCC No. 12198.

Description of the drawings:

FIG. 1 is a single crystal diffraction structure diagram of Compound 1;

FIG. 2 is a single crystal diffraction structure diagram of Compound 2;

the specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: isolation and structural characterization of Ilamycins G-R (1-12)

Fermentation of Streptomyces atratus SCSIO Zh16 Delta ilaR

(1) Preparation of seed culture medium and fermentation culture medium: each liter of culture medium contains 15g of soluble starch, 15g of soybean peptone FP410,5g of soybean flour, 15g of glycerol, 2g of CaCO3 and 30g of sea salt, the pH value is adjusted to 7.2-7.4, and the culture medium is sterilized for 30min at 115 ℃ for later use.

(2) Culturing seeds: inoculating Streptomyces atratus SCSIO ZH16 delta ilaR spores into a 5-bottle 1L triangular flask containing 200mL of seed culture medium, and fermenting at 200rpm and 28 ℃ for 30h to serve as primary seeds; inoculating the primary seed into 40L fermentation tank containing 25L fermentation medium, and fermenting at 28 deg.C under 200rpm and 20L/min sterile air and 0.1Mpa for 24 hr to obtain secondary seed culture solution.

(3) And (3) amplified fermentation of the strain: inoculating 25L of the second generation seed culture solution into a 300L fermentation tank containing 200L of fermentation medium, fermenting at 28 deg.C under 200rpm, 20L/min sterile air and 0.1Mpa for 9 days, wherein 1000g glycerol is supplemented on the 3 rd day of fermentation to obtain fermentation culture.

Extraction and separation of di-and cyclopeptide compounds

(1) Extracting the fermentation culture by adding macroporous resin into the fermentation culture, stirring overnight to adsorb the compounds in the fermentation liquid, filtering to obtain a mixture of macroporous resin and mycelium, soaking and extracting the mixture with EtOH for three times, mixing the extractive solutions, and concentrating under reduced pressure to obtain extract.

(2) Isolation of the compound: subjecting the extract to silica gel column chromatography with CHCl at different volume ratios₃Gradient elution with/MeOH (100/0,98/2,96/4,94/6,92/8,90/10,80/20,50/50v/v), collecting chloroform-methanol eluted fraction afr.3 in a volume ratio of 94: 6; AFr.3 Medium-pressure reverse-phase column (Flash C18, 40-63 μm) chromatography, MeCN-H₂O elution (volume ratio 40:60 → 80:20,0 → 130 min; 100:0,121 → 160 min; flow rate 20mL/min) was collected every 20min and divided into 8 portions (AFr) in sequence.3-1-AFr.3-8); AFr.3-3 (elution fraction at 41-60 min) high performance liquid semi-preparative column chromatography (YMC-Pack ODS-A column, 250X 20mm, 5 μm), MeCN-H₂O (volume ratio 55:45, flow rate 3.0mL/min, detection wavelength λ 220nm) to give compound 8(12.6mg, retention time 15.5min) and compound 1(19.4mg, retention time 18.1 min); AFr.3-4 (fraction eluted at 61-80 min) by silica gel column chromatography, CHCl₃MeOH elution (96: 4 by volume) according to the silica gel thin layer chromatography (CHCl as developing solvent)₃-MeOH94:6v/v) split it into two parts: AFr.3-4-1 (specific shift value between 0.4 and 0.6) and AFr.3-4-2 (specific shift value between 0.3 and 0.4), and AFr.3-4-1 was chromatographed by high performance liquid semi-preparative column (YMC-Pack ODS-A column, 250X 20mm, 5 μm), MeCN-H₂Eluting with O (volume ratio of 55:45, flow rate of 3.0mL/min, detection wavelength λ ═ 220nm) to obtain compounds 6(3.4mg, retention time of 22.1min) and 12(2.1mg, retention time of 24.2min), subjecting AFr.3-4-2 to high performance liquid chromatography (YMC-Pack ODS-A column, 250 × 20mm, 5 μm) chromatography, and subjecting to MeCN-H chromatography₂Eluting with O (volume ratio of 55:45, flow rate of 3.0mL/min, detection wavelength λ of 220nm) to obtain compounds 4(2.8mg, retention time of 20.3min) and 10(7.3mg, retention time of 23.4 min); AFr.3-5 (81-100 min elution fraction) high performance liquid semi-preparative column (YMC-Pack ODS-A column, 250X 20mm, 5 μm) chromatography, MeCN-H₂O (volume ratio 60:40, flow rate 3.0mL/min, detection wavelength λ 220nm) to give compounds 11(2.6mg, retention time 25.9min), 2(17mg, retention time 27.1 min); AFr.3-6 (elution fraction 101-₂O (volume ratio 60:40, flow rate 3.0mL/min, detection wavelength λ 220nm) to give compound 3(4.0mg, retention time 31.8 min); AFr.3-7 (elution fraction 121-₃MeOH (96: 4 by volume) and the silica gel thin layer chromatography fractions at 0.25, 0.20, 0.10 (CHCl as developing solvent) were collected₃MeOH 96:4) to give compounds 5(3.7mg), 7(13.9mg) and 9(3.2 mg).

Structural identification of Compounds 1-12

The physicochemical property data of the compounds are as follows:

ilamycin G (1) yellow crystals (C)EtOH)；[α]²⁵ _D-43(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.57),221(4.56),282(3.92)nm；¹H and¹³c nmr data are shown in table 1; (+) -HR-ESI-MS M/z1058.5576[ M + H]⁺(calcd for C₅₄H₇₆N₉O₁₃1058.5557); the single crystal diffraction structure is shown in figure 1.

Ilamycin H (2) yellow crystals (EtOH); [ alpha ] to]²⁵ _D-40(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.59),220(4.41),281(3.76)nm；¹H and¹³c nmr data are shown in table 1; (+) -HR-ESI-MS M/z1058.5554[ M + H]+(calcd for C₅₄H₇₆N₉O₁₃1058.5557); the single crystal diffraction structure is shown in figure 2.

Ilamycin I (3): yellow powder; [ alpha ] to]²⁵ _D-40(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.59),220(4.41),281(3.76)nm；¹H and¹³c nmr data are shown in table 1; (+) -HR-ESI-MS M/z 1044.5790([ M + H)]⁺,cald for C₅₄H₇₈N₉O₁₂,1044.5764)。

Ilamycin J (4) yellow powder; [ alpha ] to]²⁵ _D-20.3(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.44),219(4.38),277(3.69)nm；¹H and¹³c nmr data are shown in table 1; (+) -HR-ESI-MS M/z1042.5614([ M + H)]+,cald for C₅₄H₇₆N₉O₁₂,1042.5608)。

Ilamycin K (5) yellow powder; [ alpha ] to]²⁵ _D-8.2(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.44),219(4.38),277(3.69)nm；¹H and¹³c nmr data are shown in table 2; (+) -HR-ESI-MS M/z 1058.5549([ M + H)]+,cald for C₅₄H₇₆N₉O₁₃,1058.5557)。

Ilamycin L (6) yellow powder; [ alpha ] to]²⁵ _D-24(c 0.10,MeOH)；UV(MeOH)λmax(logε)204(4.47),220(4.41),282(3.76)nm；¹H and¹³c nmr data are shown in table 2; (+) -HR-ESI-MS M/z 1044.5782([ M + H)]+,cald for C₅₄H₇₈N₉O₁₂,1044.5764)。

Ilamycin M (7) yellow powder; [ alpha ] to]²⁵ _D-3.5(c 0.10,MeOH)；UV(MeOH)λmax(logε)201(3.89),221(3.71),282(3.14)nm；¹H and¹³c nmr data are shown in table 2; (+) -HR-ESI-MS M/z1074.5533([ M + H)]+,cald for C₅₄H₇₆N₉O₁₄,1074.5506)。

Ilamycin N (8) yellow powder; [ alpha ] to]²⁵ _D-21.0(c 0.10,MeOH)；UV(MeOH)λmax(logε)205(4.52),222(4.45),286(3.75)nm；¹H and¹³c nmr data are shown in table 2; (+) -HR-ESI-MS M/z1040.5834([ M + H)]+,cald for C₅₅H₇₈N₉O₁₁,1040.5815)。

Ilamycin O (9) yellow powder; [ alpha ] to]²⁵ _D-12.5(c 0.10,MeOH)；UV(MeOH)λmax(logε)202(4.20),223(4.03),286(3.34)nm；¹H and¹³c nmr data are shown in table 3; (+) -HR-ESI-MS M/z997.5762([ M + H)]+,cald for C₅₄H₇₇N₈O₁₀,997.5757)。

Ilamycin P (10): yellow powder; [ alpha ] to]²⁵ _D-16.8(c 0.10,MeOH)；UV(MeOH)λmax(logε)202(4.26),219(4.18),281(3.50)nm；¹H and¹³c nmr data are shown in table 3; (+) -HR-ESI-MS M/z1042.5618([ M + H)]+,cald for C₅₄H₇₆N₉O₁₂,1042.5608)。

Ilamycin Q (11) yellow powder; [ alpha ] to]²⁵ _D-53.1(c 0.10,MeOH)；UV(MeOH)λmax(logε)205(4.55),220(4.51),277(3.84)nm；¹H and¹³c nmr data are shown in table 3; (+) -HR-ESI-MS M/z1046.5382([ M + H)]+,cald for C₅₃H₇₅N₉O₁₁S,1046.5380)。

Ilamycin R (12): yellow powder; [ alpha ] to]²⁵ _D-8.9(c 0.10,MeOH)；UV(MeOH)λmax(logε)202(4.10),220(3.93),280(3.32)nm；¹H and¹³c nmr data are shown in table 3; (+) -HR-ESI-MS M/z1024.5898([ M + H)]+,cald for C₅₅H₇₈N₉O₁₀,1024.5898)。

Table 1: NMR data (700/175MHz, TMS as internal standard, ppm) for Compounds 1-4

^ameasured at 175 MHz in CD₃OD；^bmeasured at 700MHz in CD₃OD

Table 2: NMR data (700/175MHz, TMS as internal standard, ppm) for Compounds 5-8

^ameasured at 175MHz in CD₃OD；^bmeasured at 700MHz in CD₃OD；^cmeasured at 175MHz in pyrdine-d₆；^dmeasured at700MHz in pyrdine-d₆.

Table 3: NMR data (700/175MHz, TMS as internal standard, ppm) for Compounds 9-12

^ameasured at 175MHz in CD₃OD；^bmeasured at 700MHz in CD₃OD.

Through HRESIMS and nuclear magnetic resonance of the compounds and single crystal diffraction data analysis, the compounds 1-12 are all new compounds, and the structure is shown as formula I.

Example 2: antibacterial activity test analysis of compounds 1-12 shown in formula I on mycobacteria

Compounds 1-12 were tested against M.cubcumulosis H37R using a double dilution method_VThe antibacterial activity of (1). The test procedure is briefly described as follows: 1) inoculating 2ml of self-luminescence M.tubericulosis H37Rv (UAlRa) frozen at-80 ℃ into an Erlenmeyer flask containing 50ml of 7H9 (containing 0.1% twin80) culture medium, and culturing until the OD value reaches between 0.3 and 1.0; 2) preparing compounds 1-12 into 10mg/mL mother liquor by DMSO, diluting each compound to 5120-2.5 mu g/mL according to a certain proportion, taking RIF (10 mu g/mL, 1 mu g/mL) as a positive control and DMSO as a negative control, adding the corresponding compound into a 96-well enzyme label plate by 5 mu L per well, and setting three times for each concentration of each compound; 3) diluting the bacterial liquid stock solution, and taking diluted bacterial liquid with a luminous value of 3000-5000/200 mu L as bacterial liquid for detection; 4) adding 195. mu.L of diluted bacteria solution into each well of a 96-well enzyme label plate by using a calandria gun to ensure that the final concentration of each compound is 128-0.0625. mu.g/mL in sequence, culturing in a 37-degree incubator, and detecting the luminescence value at 0-7 d. The results of the activity tests showed (Table 4) that compounds 1-12 had some differences in mycobacterial activity, compounds 3, 5, 7 and 10-12 M.tuberculosis H37R_VThe activity is better; the compounds 4 and 6 show strong antitubercular activity, have the same drug effect as a positive control, and show important value in the development of antitubercular treatment drugs.

Table 4: inhibitory Activity of the substance against Mycobacterium (MIC, μ M, n ═ 3)

Claims

1. Any cyclic peptide compound represented by formula I:

2. the application of marine actinomycetes mutant strain Streptomyces atratus SCSIO Zh16 ΔilaR in preparing the described cyclic peptide compound of claim 1, and described marine actinomycetes mutant strain Streptomyces atratus SCSIO Zh16 ΔilaR is marine Streptomyces atratus SCSIO Zh16 The ilaR gene deletion mutant strain of CGMCC No. 12198.

3. a preparation method of the cyclic peptide compound 1-12 described in claim 1, is characterized in that, described compound 1-12 is from the fermentation culture of marine actinomycetes mutant Streptomyces atratus SCSIO Zh16 ΔilaR The marine actinomycete mutant strain Streptomyces atratus SCSIO Zh16 ΔilaR prepared in Streptomyces atratus SCSIO Zh16 CGMCC No.12198 is a mutant strain of ilaR gene deletion of marine Streptomyces atratus SCSIO Zh16 CGMCC No.12198.

4. preparation method according to claim 3, is characterized in that, concrete steps are as follows:

a. Preparation of marine actinomycete mutant Streptomyces atratus SCSIO Zh16 ΔilaR fermentation culture;

b. Add macroporous resin to the fermentation culture, stir to absorb the compounds in the fermentation broth, filter to obtain a mixture of macroporous resin and mycelium, the mixture is leached with EtOH, the extract is concentrated to obtain an extract, and the extract is passed through a silica gel column layer analysis, according to CHCl ₃ /MeOH gradient elution from 100/0, 98/2, 96/4, 94/6, 92/8, 90/10, 80/20, 50/50 v/v, the collection volume ratio is 94 : 6 chloroform-methanol eluted fraction Afr.3, AFr.3 was separated and purified to obtain compound 1-12.

5. preparation method according to claim 4 is characterized in that, the described fermentation culture of preparing marine actinomycete mutant Streptomyces atratus SCSIO Zh16 ΔilaR is prepared by the following method:

The marine actinomycete mutant Streptomyces atratus SCSIO Zh16 ΔilaR is inserted into the seed medium, fermented to obtain a seed culture solution, the seed culture solution is inserted into the fermentation medium, and fermented to obtain a fermented culture, the seed culture medium and fermentation Each liter of culture medium contains: 15g soluble starch, 15g soy peptone FP410, 5g soybean powder, 15g glycerol, 2g CaCO ₃ , 30g sea salt, and the pH value is adjusted to 7.2-7.4.

6. Use of any cyclic peptide compound in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-mycobacterial drug.

7. The application according to claim 6, wherein the anti-mycobacterial drug is an anti-mycobacterium tuberculosis drug.

8. An anti-mycobacterial drug, characterized in that it contains an effective amount of any cyclic peptide compound in claim 1, or a pharmaceutically acceptable salt thereof as an active ingredient.

9 . The anti-mycobacterial drug according to claim 8 , wherein the anti-mycobacterial drug is an anti-mycobacterium tuberculosis drug. 10 .