WO2017166812A1 - Rakicidins类化合物及其治疗抗致病厌氧菌疾病的用途 - Google Patents

Rakicidins类化合物及其治疗抗致病厌氧菌疾病的用途 Download PDF

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WO2017166812A1
WO2017166812A1 PCT/CN2016/104337 CN2016104337W WO2017166812A1 WO 2017166812 A1 WO2017166812 A1 WO 2017166812A1 CN 2016104337 W CN2016104337 W CN 2016104337W WO 2017166812 A1 WO2017166812 A1 WO 2017166812A1
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compound
rakicidin
ethanol
infection
pharmaceutically acceptable
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PCT/CN2016/104337
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French (fr)
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林风
江红
连云阳
周剑
陈丽
江宏磊
赵薇
陈晓明
方东升
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福建省微生物研究所
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Priority claimed from CN201610182467.1A external-priority patent/CN105753936B/zh
Priority claimed from CN201610182142.3A external-priority patent/CN105709205B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the invention relates to the field of medicine, in particular to a Rakicidins compound and the use thereof for treating diseases such as diarrhea, digestive tract inflammation, oral inflammation and skin acne caused by anaerobic bacteria.
  • Anaerobic C. difficile infection is the leading cause of antibiotic-associated diarrhea in hospitals. Metronidazole, vancomycin, and philamycin are currently recommended drugs for the treatment of C. difficile infection, but C. difficile infection in A The recurrence rate after treatment with nitrozol or vancomycin is high, and new therapeutic drugs are urgently needed. Fidamycin is a new macrolide antibiotic that can be administered orally. It was approved for the treatment of C. difficile infection in the United States and Europe in 2011.
  • Vancomycin-resistant enterococci and Pseudomonas aeruginosa and Acinetobacter baumannii have become major pathogens for surgical and wound infections, pneumonia, endocarditis, meningitis, blood-borne infections and amputations, posing a serious threat to humans. Health and life. Enterococcus (Enterococcus faecium and Enterococcus faecalis) is widely distributed in nature, often inhabited by humans, animal intestines and female genitourinary system, and is one of the normal flora of humans.
  • VRE vancomycin-resistant enterococci
  • enterococci are the important nosocomial infection pathogens after Staphylococcus, which can cause urinary tract infections, abdominal infections, pelvic inflammatory disease and endocarditis, and can cause sepsis in severe cases.
  • the case fatality rate is 21.0% to 27.5%. Therefore, the development of clinical drugs against VRE has certain prospects.
  • hypoxia is one of the characteristics of malignant solid tumors. Hypoxia is closely related to tumor angiogenesis, invasion and metastasis, radiochemotherapy resistance and poor prognosis. Hypoxia-inducible factor 1 (HIF-1) is the most critical nuclear transcriptional regulator in the regulation of hypoxia.
  • HIF-1 is selectively and continuously expressed in solid tumor tissues, and key downstream regulatory genes are closely related to the occurrence and development of tumors, such as promoting angiogenesis, cell survival, inhibiting tumor cell apoptosis, metabolic remodeling, and regulation of pH homeostasis. It is precisely because of the difference in oxygen content in the environment in which cancer cells are located in different time and space that the signal pathway that promotes tumor growth is usually not induced in normal tissues, so the hypoxia signaling pathway becomes a potential therapeutic target. Specific small molecule inhibitors targeting HIF-1 have also become the focus of research and development of tumor hypoxia-regulating drugs.
  • Rakicidin A and B have attracted attention because their 15-membered cyclolipopeptide structure contains a rare and rare octyl 4-amino-2,4-pentadienoate and has anti-tumor activity [McBrien K D, Berry R L, Lowe S E et al. Rakicidins, New Cytotoxic Lipopeptides from Micromonospora sp. Fermentation, Isolation and Characterization [J]. J Antibiot, 1995, 48: 1446].
  • the structure is as follows:
  • Rakicidin A has excellent hypoxic selective anti-tumor cell activity, and anti-colon cancer HCT-8 cell activity under hypoxic conditions is 17.5 times that under normoxic conditions [Yamazaki Y, Kunimoto S, Ikeda D .Rakicidin A: a hypoxia-selective cytotoxin [J]. Biol Pharm Bull. 2007, 30(2): 261-5.].
  • Yamauchi (2011) reported for the first time that Rakicidin A can induce apoptosis of myeloid chronic leukemia stem cells under hypoxic conditions [Takeuchi M, Ashihara E, Yamazaki Y, et al.
  • Rakicidin A practical induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells [ J]. Cancer Sci. 2011, 102(3): 591-6.]. Although the mechanism of action of this compound against hypoxic tumor cells and anti-tumor stem cells (CSC) is unclear, Rakicidin A has been considered by many peers as a promising anti-hypoxic tumor cell and anti-CSC drug.
  • Rakicidins compounds have anti-cancer activity against clinical pathogenic anaerobic bacteria, such as C. difficile, and resistance to vancomycin-resistant enterococci, which deserve further research and development.
  • Rakicidin B1 a Rakicidins compound named Rakicidin B1
  • the structural formula is as follows:
  • the compound Rakicidin B1 of the present invention is obtained by microbial fermentation, which has anti-hypoxic tumor cell activity, anti-clinical anaerobic bacteria and anti-vancomycin-resistant enterococci activity.
  • the invention discloses a strain of Micromonospora, referred to as FIM02-523, and has a classification name: Micromonospora sp. FIM02-523.
  • the deposit number of the General Microbiology Center of the China Microbial Culture Collection Management Committee is CGMCC No. 12132. Deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Deposit Date: February 18, 2016. Obtained from the soil of Putian Beach in Fujian province.
  • Rakicidin B1 can be prepared by microbial fermentation of the micromonospora strain FIM02-523.
  • the preparation method is as follows:
  • the micromonospora strain FIM02-523 with the preservation number CGMCC No.12132 was fermented, the fermentation broth was centrifuged to obtain mycelium residue, the mycelial residue was soaked with ethanol to obtain ethanol soaking solution, and the ethanol soaking solution was used for HP20 macroporous resin adsorption column.
  • Compound Rakicidin B1 White amorphous powder. Soluble in chloroform, methanol, ethanol, DMSO, insoluble in water, n-hexane, etc. Molecular weight 620.1175, molecular formula C 33 H 56 N 4 O 7 .
  • the micromonospora strain FIM02-523 can be separately prepared by microbial fermentation to obtain Rakicidin A, B, B1 and Rakicidin Bx (Rakicidin Bx is a mixture of Rakicidin B1 and Rakicidin B, and the weight ratio is about 1:4).
  • the preparation method is as follows:
  • the micromonospora strain FIM02-523 with the preservation number CGMCC No.12132 was fermented, the fermentation broth was centrifuged to obtain mycelium residue, the mycelial residue was soaked with ethanol to obtain ethanol soaking solution, and the ethanol soaking solution was used for HP20 macroporous resin adsorption column. Chromatography, The gradient was eluted with 60%-80% ethanol water to obtain eluent 1, and the eluent 1 was subjected to NM200 resin adsorption column chromatography, and eluted with a gradient of 55%-80% ethanol water to obtain an eluent 2, and the eluate was diluted with water.
  • the pharmaceutically acceptable salts of the compounds of the present invention have the same pharmacological effects as the compounds.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in the form of a conventional tablet or capsule, a sustained release tablet or capsule, a controlled release tablet or capsule, an oral solution, an injection, or the like.
  • a human dose will range from 1 mg to 5000 mg per day. Dosages outside the range may also vary depending on the dosage form and the severity of the disease.
  • Rakicidins-like compounds and Rakicidin Bx were dissolved in DMSO to 1.28 mg/ml.
  • the test concentrations were: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 ⁇ g/ml. The remaining solution of the test will be placed at -20 °C.
  • the antibiotics Fidamycin and Metronidazole were used as reference compounds.
  • Preparation of 2X solution The highest concentration was 256 ⁇ g/ml, followed by two 10-fold dilutions in 96 deep well plates to give the desired 2X solution. Dispense 100 ⁇ l to 96-well round bottom plate with a workstation. Column 12 is a negative control containing only the same volume of medium.
  • bacterial inoculum 1 day in advance (aerobic bacteria) or 2-3 days (anaerobic bacteria) on the corresponding growth plate [medium: aerobic bacteria, CAMHB (cation adjusted Mueller-Hinton medium, ion-regulated rice) Lexington's medium; anaerobic bacteria, Brucella broth supplemented with hemin (5g/mL), Vitamin K1 (1g/mL), and lysed horse blood (5% v/v)].
  • the bacteria concentration was adjusted to the day of the experiment. About 1-2 x 10 6 CFU/ml, and then transfer 100 ⁇ l to a 96-well round bottom plate (prepared compound-added 96-well plate).
  • MIC measurement For aerobic bacteria, the 96-well round bottom plate obtained above was placed at 37 ° C, 85% humidity, and cultured under atmospheric conditions for 20 hours; for anaerobic bacteria, the 96-hole round bottom plate obtained above was placed at 37 Incubate for 46-48 hours under anaerobic conditions at °C, 85% humidity. The concentration point at which bacterial growth is completely or significantly inhibited will be defined as the MIC of the compound. The results are shown in Table 1.
  • Table 1 shows that the anti-anaerobic test results show that Rakicidin A, B, B1 and Bx have good anti-anaerobic pathogenic C. difficile (including partially resistant C. difficile) activity, and the activity is comparable to that of philamycin;
  • the inhibitory activity of the infected P. acnes is stronger than that of phenazomycin and metronidazole; the activity of the clinically pathogenic anaerobic porphyromonas and S. digestive is comparable or stronger than that of phenazomycin;
  • the activity against vancomycin-resistant Enterococcus faecium (VRE) is stronger than that of philamycin, which is 4-8 times.
  • Rakicidin B1, A, B and Bx samples were dissolved in DMSO so that the solubility reached 1 mg/ml, and then diluted to a final concentration of 0.75 ug/ml, 0.5 ug/ml, 0.25 ug/ml, 0.125 ug/ml, 0.1ug/ml, 0.05ug/ml, 0.005ug/ml, 0.0025ug/ml.
  • hypoxic culture HCT-8 of human intestinal cancer cells in exponential growth period in 96-well plates (cell concentration 10 5 /ml, 100 ul / well), hypoxia ventilation for 30 minutes, close the vent valve, put Incubate at 37 ° C, incubate for 24 hr to adhere to the wall, add 100 ⁇ l/well of fresh medium with medicine, set 3 replicate wells for each concentration, and set blank control wells (with medium only) as negative control, also set 3 Double hole. After hypoxia ventilation for 30 minutes, the aeration valve was closed, placed in an incubator at 37 ° C, and the culture was continued until 72 hr, and the culture was terminated.
  • SRB assay The cultured cells will be stopped, and 10% TCA of 50% per well is added, and fixed at 4 ° C for 1 hr. Rinse with distilled water for 5 times, naturally dry, add 50 ul of 4 mg/ml SRB solution to each well, stain for 30 min at room temperature, discard the supernatant, and rinse 5 times with 1% acetic acid to remove the non-specifically bound dye. 150 ul of 10 mM Tris solution was added to each well, shaken for 5 minutes, and the OD value was measured by a microplate reader at 540 wavelength, and the inhibition rate was calculated. The IC50 value was calculated using SPSS software by converting the inhibition rate. The results are shown in Table 2.
  • the compound Rakicidin B1 of the present invention has potent antitumor activity, particularly anti-hypoxic tumor cell activity, and HCT-8 activity of hypoxic cultured tumor cells is ten times stronger than that of normoxia.
  • SD rats of Rakicidin Bx have an oral LD50 of >600 mg/kg; they are not absorbed orally.
  • mice were given drinking water containing cefoperazone sodium and intraperitoneally injected with a dose of clindamycin to change the structure of the normal intestinal flora of mice. At this time, the mice were susceptible to C. difficile.
  • the highly toxic C. difficile strain ATCC 43255 was inoculated into the digestive tract of mice by gavage. After the infection, the test compound solution was used for 7 days of treatment. The efficacy of the test compound is measured by mortality, body weight change and clinical score caused by CDI. After 7 days of treatment, continue to observe for two weeks, and evaluate the recurrence rate of CDI after stopping the drug.
  • the infected control mice began to show obvious clinical symptoms of CDI on the second day after infection (Day2), and reached the peak of death on the third day, with a survival rate of 40%.
  • Rakicidin Bx has a protective effect on C. difficile-infected mice, and the survival rate of infected mice is 80%.
  • two mice died (both mice were euthanized due to weight loss to 20.32% and 21.95%), and the surviving mice showed a downward trend in weight with mild diarrhea.
  • Surviving mice did not show up after stopping the drug Significant symptoms of CDI recurrence, and weight gradually increased, the rate of recurrent infection was 0.
  • Vancomycin had a good protective effect on mice infected with C. difficile during the 7-day treatment period.
  • piratamycin is superior to Rakicidin Bx and vancomycin.
  • the symptoms associated with CDI can be completely inhibited, but due to its narrow antibacterial spectrum and high antibacterial activity, phenoxymycin can promote the gradual recovery of intestinal flora in the test animals.
  • the recurrence rate after stopping vancomycin is as high as 100%, and the degree of infection is heavier.
  • Rakicidin Bx did not protect mice from pentamycin and vancomycin during the 7-day treatment period (inferred to be associated with lower solubility of the drug: Compound B is granular in the solvent and the actual effective dose will be less than 50 mg/ Kg), no recurrence was found in the mice after stopping the drug. This characteristic is the same as that of philamycin, and its efficacy is better than vancomycin.
  • Step 1 The fermentation culture conditions of the micromonospora strain FIM02-523 refer to the literature (Jiang Hong, Lin Ru, Zheng Wei, et al. Separation and identification of the lipopeptide compound FW523 produced by Micromonospora sphaeroides FIM02-523 Academic activity [J]. Chinese Journal of Antibiotics, 2006, 31 (5): 267-270).
  • Step 2 After the FIM02-523 fermentation broth obtained in the first step is centrifuged at 4500 rpm for 15 min, the mycelial slag is obtained, and the obtained mycelial slag is soaked twice with 2 times of anhydrous methanol or ethanol, and the alcohol-containing bacteria are used. The silk residue was again centrifuged at 4500 rpm for 15 minutes, and the supernatant was combined to obtain a fermentation extract.
  • Step 3 HP20 macroporous resin adsorption column chromatography (diameter ratio: 1:5 to 1:10, packed column volume 1.5-2.5L): the fermentation extract (40-60L) used is 50%-55% The concentration of alcohol was 40 ml/min for adsorption on the upper column; after the adsorption was completed, the gradient was eluted with alcohol at a concentration of 60%-80%, and the eluent 1 was detected by HPLC to combine the same components (30 L) containing Rakicidin B1. .
  • Step 4 Step 3: The eluent is subjected to NM200 resin adsorption column chromatography (the aspect ratio is 1:2 to 1:10, and the column volume is 1-2 L): the third eluate is 50%-55% alcohol. Concentration, flow rate is 40ml/min, adsorption on the upper column; after adsorption is complete, The ethanol was subjected to a gradient elution at a concentration of 55% to 80%, and the eluate 2 was detected by HPLC, and the same fraction (25 L) containing Rakicidin B1 was combined.
  • Step 5 Step 4 Eluent 2 was diluted with one volume of water, extracted twice with an equal volume of ethyl acetate, and concentrated under reduced pressure to give a crude material.
  • Step 6 The crude product obtained in the first step is dissolved in methanol, and subjected to medium pressure reversed phase C18 column chromatography (diameter to ratio 1:3 to 1:10) at a flow rate of 30 ml/min to a concentration of 60% to 90% acetonitrile. - Water gradient elution, fractional collection, detection of the supernatant by HPLC, and combining the same components.
  • the Micromonospora FIM02-523 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee in January 2016 under the accession number CGMCC No. 12132.
  • 13 C NMR and DEPT 135 show that the molecule contains 33 carbon signals, including 5 quaternary carbons ( ⁇ C 172.6, 172.4, 169.2, 167.6, 165.9), 4 double bond carbons [containing one sp 2 methylene carbon, two sp 2nd methyl carbon ( ⁇ C 138.4, 118.8)], 6 sp 3 methine carbons [containing two oxygenated carbon atoms ( ⁇ C 78.0, 72.4)], 13 sp 3 methylene carbons and 5 Methyl carbon atom ( ⁇ C 36.5, 19.1, 15.4, 13.2, 11.2).
  • H-2 is related to C-1/C-5
  • H-3 is related to C-4
  • OH-3 is related to C-2/C-3
  • H 3 -7 is related to C-6/C-8
  • H-9 is related to C-8/C-11
  • H-10 is related to C-8/C-11
  • Hb -12 is related to C-10/C-11
  • H-15 is related to C-1
  • H 3 -30 is related to C-28/C-29
  • H 3 -32 is related to C-15/C-16/C-17
  • H 3 -33 is related to C-27/C-28/C-29.
  • the chemical shifts of hydrogen and carbon are listed in the table below. :

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Abstract

本申请公开了一种Rakicidins类化合物，Rakicidin B1。本申请还公开了Rakicidin A、B、B1在制备治疗厌氧菌感染的药物中的用途。

Description

Rakicidins类化合物及其治疗抗致病厌氧菌疾病的用途 技术领域
本发明涉及医药领域，具体涉及一种Rakicidins类化合物及其治疗厌氧菌引起的腹泻、消化道炎症、口腔炎症及皮肤痤疮等疾病的用途。
背景技术
厌氧的艰难梭菌感染是医院内抗生素相关性腹泻的首要病因，甲硝唑、万古霉素、菲达霉素是目前用于艰难梭菌感染治疗的推荐药物，但艰难梭菌感染在甲硝唑或万古霉素治疗后的复发率均很高，迫切需要新的治疗药物。菲达霉素是一种可口服给药的新型大环内酯类抗生素，2011年在美国和欧洲获准用于治疗艰难梭菌感染。耐万古霉素肠球菌以及铜绿假单胞菌和鲍曼不动杆菌已成为手术和伤口感染、肺炎、心内膜炎、脑膜炎、血源感染和截肢的主要致病菌，严重威胁着人类的健康和生命。肠球菌(屎肠球菌和粪肠球菌)广泛分布在自然界，常栖居人、动物的肠道和女性泌尿生殖系统，是人类的正常菌群之一。近年来，由于抗菌药物的广泛应用，使原本就对β-内酰胺类、氨基糖苷类抗菌药物具有内在抗药性的肠球菌耐药性进一步扩大，逐渐形成了多重耐药菌。在我国，耐万古霉素肠球菌(VRE)感染的发生率呈逐年上升趋势，VRE已成为医院感染的重要病原菌之一。在需氧革兰氏阳性球菌中，肠球菌是仅次于葡萄球菌的重要院内感染致病菌，可引起泌尿道感染、腹腔感染、盆腔炎和心内膜炎，严重时可导致脓毒症，病死率达21.0％～27.5％。因此开发抗VRE的临床药物具有一定前景。
抗肿瘤药物市场近年来高速增长，2014年抗癌药市场全球销售额1000亿美元；到2018年，抗癌药市场将达到1470亿美元，复合增长率11.6％，研发抗癌药将获利巨大。缺氧是恶性实体肿瘤的特征之一，缺氧与肿瘤的血管新生、侵袭转移、放化疗抵抗和预后不良等密切相关。低氧诱导因子1(hypoxia-inducible factor 1,HIF-1)是缺氧效应调控中最为关键的核转录调控因子。HIF-1在实体肿瘤组织内选择性持续高表达,下游关键调控基因与肿瘤的发生发展密切相关,如促进血管生成、细胞存活、抑制肿瘤细胞凋亡、代谢重塑以及pH稳态的调节。正是由于不同时空下癌症细胞所处的环境中氧含量的不同，由此激活的促进肿瘤生长的信号通路通常不会在正常组织中被诱导，所以乏氧信号通路成为潜在的治疗靶点。以HIF-1为靶点的特异性小分子抑制剂也成为肿瘤缺氧效应调控药物研发的重点。
Rakicidin A和B因为其15元环脂肽结构中含1个稀有罕见的4-氨基-2,4-戊二烯酸辛酯并有抗肿瘤细胞活性而受到关注[McBrien K D,Berry R L,Lowe S E et al.Rakicidins,New Cytotoxic Lipopeptides from Micromonospora sp.Fermentation,Isolation and Characterization[J].J Antibiot,1995,48:1446]。结构式如下:
Figure PCTCN2016104337-appb-000001
Yamazaki(2007)研究发现Rakicidin A具有卓越的乏氧选择性抗肿瘤细胞活性，在乏氧条件下抗结肠癌HCT-8细胞活性是常氧条件下的17.5倍[Yamazaki Y,Kunimoto S,Ikeda D.Rakicidin A:a hypoxia-selective cytotoxin[J].Biol Pharm Bull.2007,30(2):261-5.]。Takeuchi(2011)首次报道Rakicidin A乏氧条件下能诱导髓性慢性白血病干细胞的凋亡[Takeuchi M,Ashihara E,Yamazaki Y,et al.Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells[J].Cancer Sci.2011,102(3):591-6.]。该化合物抗乏氧肿瘤细胞及抗肿瘤干细胞(CSC)的作用机制虽不清楚，但Rakicidin A已被许多同行认为是一个极富开发前景的抗乏氧肿瘤细胞及抗CSC药物。
发明人课题组于2006年在国内首先报道从微生物代谢产物中分离到Rakicidin A和B，并对Rakicidin B(FW523-3)的有关生物学活性进行了初步研究(文献1；江红，林如，程元荣.海洋小单孢菌来源的轻快菌素B FW523-3的体外抗肿瘤活性.中国抗生素杂志,2008,33(9):531；文献2；Xie JJ,Zhou F,Li EM,Jiang H,Du ZP,Lin R,Fang DS,Xu LY.FW523-3,a novel lipopeptide compound,induces apoptosis in cancer cells.Mol Med Rep.2011，4(4):759-63)。
国内外有关RakicidinA、B的活性的报道仅见上述的抗肿瘤细胞及抗肿瘤干细胞活性，其他生物学活性未见报道。
发明内容
我们首次发现Rakicidins类化合物具有抗临床致病厌氧菌艰难梭菌等、抗耐万古霉素肠球菌等活性，值得进一步研发。
本发明还公开了一个Rakicidins类化合物，命名为Rakicidin B1，结构式如下：
Figure PCTCN2016104337-appb-000002
本发明的化合物RakicidinB1通过微生物发酵获得，其具有抗乏氧肿瘤细胞活性、抗临床致病厌氧菌和抗耐万古霉素肠球菌活性。
本发明公开了一个小单孢菌菌株，简称：FIM02-523，分类命名：Micromonospora sp.FIM02-523.其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为：CGMCC No.12132。保藏地址：北京市朝阳区北辰西路1号院3号。保藏日：2016年2月18日。从福建省莆田海滩土中获得。
小单孢菌株FIM02-523通过微生物发酵可以制备得到RakicidinB1。制备方法如下：
将保藏编号为CGMCC No.12132的小单孢菌菌株FIM02-523进行发酵，发酵液离心，获得菌丝渣，菌丝渣用乙醇浸泡得到乙醇浸泡液，乙醇浸泡液进行HP20大孔树脂吸附柱层析，60％-80％乙醇水梯度洗脱，得到洗脱液1，洗脱液1进行NM200树脂吸附柱层析，55％-80％乙醇水梯度洗脱得到洗脱液2，洗脱液用水稀释后，用乙酸乙酯萃取，浓缩获得粗品用甲醇溶解，进行中压反相C18柱层析，60-90％乙腈水梯度洗脱，分部收集，用HPLC检测收集液，即得。
化合物Rakicidin B1:白色无定型粉末。溶于氯仿、甲醇、乙醇、DMSO，不溶于水、正己烷等。分子量620.1175，分子式C33H56N4O7。
小单孢菌株FIM02-523通过微生物发酵可以分别制备获得RakicidinA、B、B1和RakicidinBx(RakicidinBx为RakicidinB1和RakicidinB的混合物，重量比例为1:4左右)。制备方法如下：
将保藏编号为CGMCC No.12132的小单孢菌菌株FIM02-523进行发酵，发酵液离心，获得菌丝渣，菌丝渣用乙醇浸泡得到乙醇浸泡液，乙醇浸泡液进行HP20大孔树脂吸附柱层析， 60％-80％乙醇水梯度洗脱，得到洗脱液1，洗脱液1进行NM200树脂吸附柱层析，55％-80％乙醇水梯度洗脱得到洗脱液2，洗脱液用水稀释后，用乙酸乙酯萃取，浓缩获得粗品用甲醇溶解，进行中压反相C18柱层析，60-90％乙腈水梯度洗脱，分部收集，用HPLC检测收集液，合并相同组分即分别得到馏分A、B1、B和Bx，浓缩至干即分别获得纯品RakicidinA、B1、B以及RakicidinBx。
本发明的化合物其药学上可接受的盐与化合物具有同样的药理功效。
本发明还提供了一种药物组合物，其中含有本发明化合物和药学上可接受的载体。所述药物组合物可以是普通片剂或胶囊、缓释片剂或胶囊、控释片剂或胶囊、口服液、注射剂等制剂学上常规的制剂形式。
一般地，本发明化合物用于治疗时，人用剂量范围为1mg～5000mg/天。也可根据剂型的不同和疾病严重程度，使用剂量超出该范围。
药效学实验证明，Rakicidins类化合物具有良好的抗临床致病厌氧菌功效，下面是部分试验及结果：
一、抗致病厌氧菌活性测试
将上述3个Rakicidins类化合物及RakicidinBx在DMSO中溶解成1.28mg/ml。其测试浓度为：128，64，32，16，8，4，2，1，0.5，0.25，0.125μg/ml。测试所剩溶液将放置于-20℃。抗生素非达霉素和甲硝唑作为参照化合物。2×溶液的配制：最高浓度为256μg/ml，紧接着在96深孔板中经过10次的两倍稀释，得到所需的2×溶液。用工作站分装100μl到96孔圆底板。第12列是阴性对照，仅含相同体积的培养基。
细菌接种物的准备：提前1天(好氧菌)或2-3天(厌氧菌)在相应生长平板上[培养基:好氧菌,CAMHB(cation adjusted Mueller-Hinton medium，离子调节的米勒辛顿培养基；厌氧菌,Brucella broth supplemented with hemin(5g/mL),Vitamin K1(1g/mL),and lysed horse blood(5％v/v)]接种。实验当天将细菌浓度调到约1-2×106CFU/ml，然后转移100μl到96-孔圆底板中(中准备的加有化合物的96孔板)。
MIC测定：对于好氧菌，将以上得到的96-孔圆底板放置于37℃，85％湿度，大气条件下培养20小时；对于厌氧菌，将以上得到的96-孔圆底板放置于37℃，85％湿度条件下，厌氧条件下培养46-48小时。细菌生长被完全或明显抑制的浓度点将被定义为该化合物的MIC。结果见表1.
表1Rakicidins抗菌活性
Figure PCTCN2016104337-appb-000003
表1可见，抗厌氧菌测试结果表明RakicidinA、B、B1及Bx具有良好的抗厌氧致病的艰难梭菌(包括部分耐药的艰难梭菌)活性，活性与菲达霉素相当；对引起皮肤感染的痤疮丙酸杆菌的抑制活性强于菲达霉素和甲硝唑；对临床致病的厌氧牙龈卟啉单孢菌、消化链球菌的活性均与菲达霉素相当或更强；对耐万古霉素屎肠球菌(VRE)的活性均强于菲达霉素，是其4-8倍。
二、抗肿瘤活性测试
分别将Rakicidin B1、A、B和Bx样品溶解在DMSO中使得溶度达到1mg/ml，再分别稀释使得终浓度为0.75ug/ml、0.5ug/ml、0.25ug/ml、0.125ug/ml、0.1ug/ml、0.05ug/ml、 0.005ug/ml、0.0025ug/ml。
普通培养：取处于指数增长期的人肠癌细胞HCT-8等分别种在96孔板里(细胞浓度为105个/ml，100ul/孔)，培养24hr使其贴壁，加100ul/孔带药新鲜培养基,每个浓度设3个复孔，并设空白对照孔(只加培养基)作为阴性对照，同样设3个复孔。继续培养至72hr，终止培养。
乏氧培养：取处于指数增长期的人肠癌细胞HCT-8种在96孔板里(细胞浓度为105个/ml，100ul/孔)，乏氧通气30分钟，关闭通气阀门，放入培养箱37℃，培养24hr使其贴壁，加100ul/孔带药新鲜培养基,每个浓度设3个复孔，并设空白对照孔(只加培养基)作为阴性对照，同样设3个复孔。乏氧通气30分钟，关闭通气阀门，放入培养箱37℃，继续培养至72hr，终止培养。
SRB检测：将终止培养的细胞，每孔加10％TCA 50ul，4℃条件固定1hr。用蒸馏水冲洗5遍，自然晾干后每孔加入4mg/ml SRB溶液50ul，室温下染色30min，弃上清，用1％乙酸冲洗5遍以去除非特异性结合的染料。每孔加入150ul 10mM Tris溶液，震荡5分钟，在540波长下酶标仪测OD值，并计算抑制率。通过对抑制率的换算,使用SPSS软件计算出IC50值。结果见表2.
Figure PCTCN2016104337-appb-000004
表2RakicidinB1的乏氧肿瘤细胞毒性
Figure PCTCN2016104337-appb-000005
上述结果表明本发明化合物Rakicidin B1等具有强效抗肿瘤活性特别是抗乏氧肿瘤细胞活性，且对乏氧培养的肿瘤细胞HCT-8活性较常氧的强十几倍。
三、毒性药代
RakicidinBx的SD大鼠口服LD50>600mg/kg；口服不吸收。
体内活性
测试RakicidinBx在小鼠艰难梭菌感染(Clostridium difficile infection,CDI)模型中的体 内药效，并与万古霉素和非达霉素比较。
1、方法：给小鼠饮用含头孢哌酮钠的饮用水加上腹腔注射一个剂量的克林霉素以改变小鼠正常肠道菌群的结构，此时小鼠对艰难梭菌易感。高毒艰难梭菌菌株ATCC 43255通过灌胃的方式接种到小鼠的消化道。感染后使用待测化合物溶液进行为期7天的治疗。通过CDI造成的死亡率，体重变化和临床评分来测评待测化合物的药效。7天治疗结束后，继续观察两周，测评停药后的CDI复发率。
2、表3治疗方案
Figure PCTCN2016104337-appb-000006
注：p.o.，灌胃；q.d.，一天一次给药；mpk，mg/kg。
3、结果见表4。
表4存活率和复发率
Figure PCTCN2016104337-appb-000007
4、感染对照组小鼠在感染后的第二天(Day2)开始出现明显的CDI关联临床症状，第三天达到死亡高峰，存活率为40％。RakicidinBx对艰难梭菌感染小鼠有一定的保护作用，被感染小鼠存活率为80％。在治疗期间，两只小鼠死亡(两只小鼠均因体重降至20.32％和21.95％被处以安乐死)，幸存小鼠体重呈下降趋势，并伴有轻度腹泻。存活的小鼠在停药后未显示出 明显的CDI复发症状，并且体重呈缓慢上升趋势，复发感染率为0。万古霉素在7天的治疗期内，对艰难梭菌感染的小鼠有较好的保护作用，组内无小鼠死亡，体重无明显下降，无明显肠炎症状。CDI复发在万古霉素停药后的第四天(Day10)出现，复发感染期间两只小鼠出现死亡(一只为自然死亡；另一只体重降至23％，执行安乐死)，其余存活小鼠体重下降及腹泻程度较重，复发率高达100％。菲达霉素组内的小鼠在给药期间未见明显的感染症状，体重呈缓慢上升趋势。停药后，Day9到Day13天，组内小鼠体重有所下降，但期间并未观察到粘便、腹泻等感染相关症状，复发感染率为0。
5、结论
在艰难梭菌感染模型中，菲达霉素的药效优于Rakicidin Bx与万古霉素。在7天的治疗期，与万古霉素一样都能够完全抑制CDI关联的症状，但菲达霉素因其抗菌谱较窄且抗菌活性较高，能促进受试动物肠道菌群状态逐步恢复平衡，从而避免停药后的复发感染。而万古霉素停药后的复发率高达100％，且感染程度较重。Rakicidin Bx在7天治疗期间对小鼠的保护作用不及菲达霉素和万古霉素(推断与该药物溶解度较低有关：化合物B在溶剂中呈颗粒状，实际起效剂量会低于50mg/kg)，停药后组内小鼠未见复发，该特性与菲达霉素相同，药效优于万古霉素。
具体实施方式
实施例1
RakicidinB1的制备
步骤一：小单孢菌菌株FIM02-523的发酵培养条件参照文献(江红，林如，郑卫，等.海洋青铜小单孢菌FIM02-523产生的脂肽类化合物FW523的分离鉴别和生物学活性[J].中国抗生素杂志,2006,31(5):267-270)。
步骤二：将步骤一所得的FIM02-523发酵液通过4500rpm离心15min后，获得菌丝渣，把获得的菌丝渣用2倍体积的无水甲醇或乙醇过夜浸泡2次，将含有酒精的菌丝渣再次以4500rpm离心15min后将上清液合并，得到发酵提取液。
步骤三：HP20大孔树脂吸附柱层析(径高比为1:5～1:10，装柱体积1.5-2.5L)：采用的发酵提取液(40-60L)以50％-55％的酒精浓度，流速为40ml/min进行上柱吸附；吸附完全后，以浓度60％-80％的酒精进行梯度洗脱，用HPLC检测洗脱液1，合并含有Rakicidin B1的相同组分(30L)。
步骤四：步骤三洗脱液进行NM200树脂吸附柱层析(径高比为1:2～1:10，装柱体积1-2L)：将步骤三洗脱液以50％-55％的酒精浓度，流速为40ml/min，进行上柱吸附；吸附完全后，以 浓度55％-80％的酒精进行梯度洗脱，用HPLC检测洗脱液2，合并含有Rakicidin B1的相同组分(25L)。
步骤五：步骤四洗脱液2经过一倍体积水稀释后，用等体积乙酸乙酯萃取2次，减压浓缩，得粗品。
步骤六：将步骤五获得的粗品，用甲醇溶解，进行中压反相C18柱层析(径高比1:3～1:10)，流速30ml/min，以浓度为60％-90％乙腈-水梯度洗脱，分部收集，用HPLC检测收集液，合并相同组分。
所述的小单孢菌FIM02-523于2016年1月藏与中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.12132。
化合物Rakicidin B1:白色无定型粉末。溶于氯仿、甲醇、DMSO，不溶于水。高分辨质谱(HR-ESI-MS)显示其分子离子峰[M+H]+为621.1175，推断其分子式为C33H56N4O7，不饱和度为8。13CNMR和DEPT135显示该分子含有33个碳信号，包括5个季碳(δC 172.6,172.4,169.2,167.6,165.9)，4个双键碳[包含一个sp2亚甲基碳，两个sp2次甲基碳(δC 138.4,118.8)]，6个sp3次甲基碳[包含两个连氧碳原子(δC 78.0,72.4)]，13个sp3亚甲基碳和5个甲基碳原子(δC 36.5,19.1,15.4,13.2,11.2)。1HNMR显示该化合物有4个双键质子[δH 6.87(1H,d,J＝15.0Hz),6.16(1H,d,J＝15.0Hz),5.44(1H,s),5.32(1H,s)],5个可交换质子[δH 8.88(1H,s),8.05(1H,d,J＝9.9Hz),7.31(1H,s),7.28(1H,s),5.66(1H,d,J＝6.0Hz)],5个甲基质子[δH 2.95(3H,s),1.05(3H,d,J＝7.0Hz),0.93(3H,d,J＝6.9Hz),0.83(3H,t,J＝7.0Hz),0.81(3H,d,J＝6.7Hz)]。所有的氢质子通过HSQC谱1H–13C相关进行指认。1H–1H COSY相关谱和质子的耦合常数显示该化合物含有4个独立的自旋耦合体系：NH-2–C-2–C-3–OH-3,C-9–C-10,C-31–C-14–C-15–C-16(C-32)–C-17–C-18和C-26–C-27–C-28(C-33)–C-29–C-30。结合1H–1H COSY和HMBC可知，H-2与C-1/C-5相关，H-3与C-4相关，OH-3与C-2/C-3相关，Ha-6与C-5/C-7相关，H3-7与C-6/C-8相关，H-9与C-8/C-11相关，H-10与C-8/C-11相关，Hb-12与C-10/C-11相关，H-15与C-1相关，H3-30与C-28/C-29相关，H3-31与C-13/C-14/C-15相关，H3-32与C-15/C-16/C-17相关，以及H3-33与C-27/C-28/C-29相关，氢和碳的化学位移列于下表：
Figure PCTCN2016104337-appb-000008
Figure PCTCN2016104337-appb-000009

Claims (8)

  1. 结构式(I)的化合物或其药学上可接受的盐：
    Figure PCTCN2016104337-appb-100001
  2. 权利要求1的化合物的制备方法，包括：将保藏编号为CGMCC No.12132的小单孢菌菌株FIM02-523进行发酵，发酵液离心，获得菌丝渣，菌丝渣用乙醇浸泡得到乙醇浸泡液，乙醇浸泡液进行HP20大孔树脂吸附柱层析，60％-80％乙醇水梯度洗脱，得到洗脱液1，洗脱液1进行NM200树脂吸附柱层析，55％-80％乙醇水梯度洗脱得到洗脱液2，洗脱液用水稀释后，用乙酸乙酯萃取，浓缩获得粗品用甲醇溶解，进行中压反相C18柱层析，60-90％乙腈水梯度洗脱，分部收集，用HPLC检测收集液，即得。
  3. 一种药物组合物，其中含有权利要求1的化合物或其药学上可接受的盐及药学上可接受的载体。
  4. 下列任一结构的化合物或其药学上可接受的盐用于制备治疗致病厌氧菌感染的疾病的药物的用途：
    Figure PCTCN2016104337-appb-100002
    Figure PCTCN2016104337-appb-100003
  5. 权利要求4的用途，其中致病厌氧菌感染的疾病是艰难梭菌感染的疾病。
  6. 权利要求5的用途，其中艰难梭菌感染的疾病是艰难梭菌感染引起的腹泻、肠炎、消化道感染、口腔感染或皮肤痤疮。
  7. 下列任一结构的化合物或其药学上可接受的盐用于制备抗耐万古霉素肠球菌的药物的用途。
    Figure PCTCN2016104337-appb-100004
  8. 一种小单孢菌菌株，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏号为：CGMCC No.12132。
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CN110698541A (zh) * 2019-08-12 2020-01-17 福建省微生物研究所 一种天然Rakicidins类化合物Rakicidin J及其发酵提取方法
CN110698537A (zh) * 2019-08-12 2020-01-17 福建省微生物研究所 一种天然Rakicidins类化合物Rakicidin B1-2及其发酵提取方法
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CN111116707A (zh) * 2019-12-20 2020-05-08 福建省微生物研究所 一种Rakicidins氨基甲酸酯类衍生物及其制备方法与应用
CN113527416A (zh) * 2021-08-11 2021-10-22 南开大学 一种硝基还原酶响应氨基酸及肿瘤乏氧荧光探针的制备方法

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