CN111116707A - Rakicidins carbamate derivative and preparation method and application thereof - Google Patents
Rakicidins carbamate derivative and preparation method and application thereof Download PDFInfo
- Publication number
- CN111116707A CN111116707A CN201911325504.XA CN201911325504A CN111116707A CN 111116707 A CN111116707 A CN 111116707A CN 201911325504 A CN201911325504 A CN 201911325504A CN 111116707 A CN111116707 A CN 111116707A
- Authority
- CN
- China
- Prior art keywords
- rakicidins
- rakicidin
- carbamate
- compound
- pharmaceutically acceptable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medical chemistry, and particularly relates to a Rakicidins carbamate derivative, and further discloses a preparation method and application thereof. According to the Rakicidins carbamate derivative, modification and optimization are carried out on the structural basis of the Rakicidins carbamate derivative through a semisynthetic modification method, esterification and amination reaction are carried out on the 3-position of a known Rakicidins compound, different carbamates are connected to the structure of the Rakicidins compound, the structural modification of the Rakicidins compound is realized, the maximum cytotoxicity is reduced as far as possible on the basis of retaining the activity of potential clostridium difficile resistance, and the Rakicidins carbamate derivative serving as an anti-clostridium difficile active compound with a brand new structural type has the potential development value further improved.
Description
Technical Field
The invention belongs to the technical field of medical chemistry, and particularly relates to a Rakicidins carbamate derivative, and further discloses a preparation method and application thereof.
Background
Clostridium Difficile (CD) is the leading cause of antibiotic-associated diarrhea and colitis, and the incidence and severity of disease of Clostridium difficile-associated diarrhea (CDAD) has been on the rise worldwide since 2003, especially the emergence and outbreak of the high-producing strain c.difficile (BI/NAPI/027), making CD one of the leading causative bacteria of hospital-and community-acquired diarrhea. Clostridium Difficile Infection (CDI) has also become one of the clinically high mortality infections.
At present, the clinical treatment of CDI is not a few, and the common medicines mainly comprise metronidazole, vancomycin, phenanthromycin and the like. However, the treatment effect of vancomycin and metronidazole on severe cases is limited, the recurrence rate is up to more than 25% -60%, and the metronidazole drug resistance condition is not optimistic; although the curative effect of the fidaxomicin on acute CDAD infection is equivalent to that of vancomycin and the recurrence rate is low, the recurrence rate of the fidaxomicin for epidemic infectious diseases including clostridium difficile virulent strain NAPI/BI/027 is still high (24 percent), the overall effect is not better than that of the fidaxomicin, and the recurrence rate of the treatment on CDAD recurrence cases also reaches 20 percent. Therefore, more treatments must be sought in the face of these ever-increasing threats of infection and the continuing risk of recurrence.
Rakicidin sA-I and B1 series derivatives are respectively separated from Micromonospora and Streptomyces fermentation liquids, and the series of compounds are all detected to have inhibitory activity on different tumor cell strains or certain special bacteria. Wherein, Rakicidins A/B/E/G/H/I are all produced from micromonospora, Rakicidins C/D/F are produced from streptomyces, and the source range of producing bacteria is wider from land to sea. Structural commonalities of Rakicidins are: contains the rare characteristic 4-amino-2, 4 pentadiene amide (APDA), Sarcosine (Sarcosine, SAR), and lipophilic long fatty chain structure; wherein Rakicidins C and F are different from A-D, E-I in that Glutamine (GLN) is used for replacing Asparagine (Asparagine, ASN) outside the ring; differences in the structure of the long fat chain also determine differences between homologs.
Research shows that Rakicidins can obviously inhibit the proliferation of various cancer cells under the condition of normoxic culture in vitro, and particularly has strong inhibitory activity on high-metastatic, drug-resistant and refractory cancer cells of some people; has strong selective cytotoxicity on tumor hypoxic cells, and Rakicidin A has particularly obvious inhibition effect on HCT-8 and PANC-1 tumor cell strains under hypoxic conditions, and also has obvious activity on chronic myelocytic leukemia tumor stem cell CML. In addition, Rakicidins have broad-spectrum anti-tumor cell effects and also have good inhibitory activity on partial anaerobes.
When the research group detects the activity of Rakicidins compounds, the substances have different degrees of bactericidal activity to gram-positive anaerobic bacteria such as clostridium difficile, anaerobic digestion streptococcus, porphyromonas gingivalis, propionibacterium acnes and the like, and the MIC values are mostly between 0.125 and 0.25 mu g/mL and are equivalent to or even stronger than the activity of control metronidazole. Such compounds are expected to have utility in the treatment of vancomycin-resistant enterococcus faecium infections, clostridium difficile infections, digestive tract infections, gingival infections, facial acne infections, and the like. However, the Rakicidins compounds still have certain obstacles in application due to the unstable physicochemical properties and poor solubility of the skeleton structure. Therefore, those skilled in the art are fully expected to have a more clinically useful Rakicidin product to provide a more ideal choice for clinical treatment of clostridium difficile.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a Rakicidins carbamate derivative which has strong bacteriostatic activity on various clostridium difficile resistant bacteria and obviously reduced cytotoxicity;
the second technical problem to be solved by the invention is a preparation method and application of the Rakicidins carbamate derivatives.
In order to solve the technical problems, the Rakicidins carbamate derivative and the pharmaceutically acceptable salt, isomer or ester thereof are disclosed by the invention, the derivative is obtained by esterification and amination reaction of a Rakicidins compound, and the Rakicidins carbamate derivative has a structure shown as the following formula (I):
in the formula (I), the compound is shown in the specification,
the R isaAnd RbIndependently of each other, is selected from hydrogen, amino, C1-C10 alkyl, C3-C7 cycloalkyl, C2-C10 alkenyl or C2-C10 alkynyl; alternatively, the first and second electrodes may be,
the R isaAnd RbTogether with the nitrogen atom form a 5-to 10-membered heterocyclic group, other than RaAnd Rb0-4 heteroatoms selected from N, O and S in addition to the nitrogen atom to which it is attached;
the Rakicidins compounds include Rakicidin A, Rakicidin B1, Rakicidin E, Rakicidin G, Rakicidin H or Rakicidin I.
Preferably, in the formula, R isaAnd RbIndependently of each other, is selected from hydrogen, C1-C10 alkyl, C3-C7 cycloalkyl, C2-C10 alkenyl, C2-C10 alkynyl; or, said RaAnd RbTogether with the nitrogen atom form a 5-to 10-membered heterocyclic group, other than RaAnd RbThe nitrogen atom to which it is attached contains 0 to 2 hetero atoms selected from N and O;
more preferably, in the formula (II), R isaAnd RbIndependently of one another, are selected from hydrogen, C1-C5 alkyl, C3-C6 cycloalkyl; or, said RaAnd RbTogether with the nitrogen atom form a 5-to 7-membered heterocyclic group, other than RaAnd RbThe nitrogen atom to which it is attached contains 0 to 2 hetero atoms selected from N and O.
Specifically, the Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof are selected from the following compounds:
Rakicidin-3-O- (diethyl carbamate);
Rakicidin-3-O- (morphininyl-4-carbonyl formate);
Rakicidin-3-O- (1-methylpiperidine-4-carbamate);
Rakicidin-3-O- (4-hydroxypiperidinyl-1-carboxylate).
More preferably, said Rakicidins compound is selected from Rakicidin B1.
The invention also discloses a method for preparing the Rakicidins carbamate derivatives, which comprises the following steps:
(1) dissolving a Rakicidins compound with a selected structure in an organic solvent, and adding a chloroformate compound with a selected structure for reaction to obtain an esterification reactant of the Rakicidins compound;
(2) and adding the obtained esterification reactant into organic amine with a selected structure for amination reaction to obtain the Rakicidins carbamate derivative with the required structure.
Specifically, in the preparation method of the Rakicidins carbamate derivative:
in the step (1), the reaction temperature is controlled to be-10-0 ℃;
in the step (2), the reaction temperature is controlled to be-5 DEG C
Specifically, the organic solvent includes pyridine.
The invention also discloses application of the Rakicidins carbamate derivative and pharmaceutically acceptable salts, isomers or esters thereof in preparation of clostridium difficile resistant medicines.
In particular, the resistance to clostridium difficile comprises the prevention and/or treatment of diarrhea and/or cancer associated with clostridium difficile. The related diseases are selected from: colonic infections, occur in patients who have recently used antibiotics to disrupt the normal intestinal flora, pseudomembranous colitis, Clostridium Difficile Infection (CDI) or Clostridium Difficile Associated Diarrhea (CDAD).
The invention also discloses a medicine for resisting clostridium difficile, which is prepared by taking the Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof as active ingredients and adding a pharmaceutically acceptable carrier.
The invention also discloses application of the Rakicidins carbamate derivative and pharmaceutically acceptable salts, isomers or esters thereof in preparing medicaments with antitumor activity.
The invention also discloses a medicament with anti-tumor activity, which comprises the Rakicidins carbamate derivatives, pharmaceutically acceptable salts, isomers or esters thereof as active ingredients, and pharmaceutically acceptable carriers.
In the method for preventing and/or treating clostridium difficile-related diseases and/or cancers, the Rakicidins carbamate derivative is administered to a subject in need thereof in a prevention and/or treatment effective amount.
According to the Rakicidins carbamate derivative, modification and optimization are carried out on the structural basis of the Rakicidins carbamate derivative through a semisynthetic modification method, esterification and amination reaction are carried out on the 3-position of a known Rakicidins compound, different carbamates are connected to the structure of the Rakicidins compound, the structural modification of the Rakicidins compound is realized, the maximum cytotoxicity is reduced as far as possible on the basis of retaining the activity of potential clostridium difficile resistance, and the Rakicidins carbamate derivative serving as an anti-clostridium difficile active compound with a brand new structural type has the potential development value further improved.
According to the scheme, the carbamate derivative of Rakicidin B1 is taken as an example, the in vitro activity of the derivative shows that the derivative has good clostridium difficile resistance activity, and tumor cells A549 and HT-29 and tumor cells Caco-2 under normoxic and hypoxic culture have certain inhibition effects, and the derivative can be used as a novel clostridium difficile resistance medicament and/or an information anticancer medicament for development and utilization.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 is a cytotoxicity study of Rakicidin B1 and its derivatives against tumor cells A549 and HT-29 under normoxic conditions;
FIG. 2 shows the results of cytotoxicity studies of compounds Rakicidin B1 and 3B against Caco-2 tumor cells under normoxic and hypoxic conditions;
FIG. 3 shows the results of a selective study of the cytotoxicity of the compounds Rakicidin B1 and 3B against Caco-2 tumor cells under normoxic and hypoxic conditions.
Detailed Description
In the Rakicidins carbamate derivatives, the groups such as C1-C4 alkyl, C1-4 alkyl, C1-C4 alkyl and the like in optional groups have the same meaning and all represent straight-chain or branched alkyl with 1-4 carbon atoms. Other situations may be similarly understood. In the context of the present invention, the group "C1-4 alkyl" includes that which is stated individually and in combination with other groups, and may be chosen, for example, from C1-3 alkyl, C1-2 alkyl. Likewise, C1-4 alkoxy may be selected from C1-3 alkoxy, C1-2 alkoxy, for example.
In addition, the compounds of the present invention are essentially derivatives of Rakicidins compounds substituted by the hydroxyl at the C3 position, so that the names can be expressed by the substituents on the hydroxyl at the C3 position, still based on the Rakicidins parent nucleus, and the names of the derivatives as described in the specification are named as follows.
In the following embodiments of the present invention, the preparation method of the Rakicidins carbamate derivatives comprises the following steps:
(1) dissolving a Rakicidins compound with a selected structure in an organic solvent, and adding a chloroformate compound with a selected structure for reaction to obtain an esterification reactant of the Rakicidins compound;
(2) and adding the obtained esterification reactant into organic amine with a selected structure for amination reaction to obtain the Rakicidins carbamate derivative with the required structure.
It should be noted that, in the method for synthesizing the Rakicidins carbamate derivatives of the present invention, various raw materials used in the reaction can be prepared by those skilled in the art according to the prior knowledge, or can be prepared by methods known in the literature or methods known by those skilled in the art of organic chemistry, or can be commercially available. The intermediates, starting materials, reagents, reaction conditions, etc. used in the above reaction schemes may be appropriately modified according to the knowledge of those skilled in the art.
In the following examples of the invention, NMR spectra of the prepared compounds were determined using BrukeraRx-300 and mass spectra were determined using Agilent 1100 LC/MSD; all reagents used were analytically or chemically pure.
Examples 1 to 4
The corresponding Rakicidin carbamate derivatives were synthesized as exemplified by the Rakicidin B1 compound in examples 1-4 below, according to the following equation:
the compound Rakicidin B1(0.5g, 0.8mmol) was dissolved in 10.0mL pyridine solution, p-nitrophenyl chloroformate (1.0g, 5mmol) was added with stirring at-5 deg.C and after about 0.5 hour of reaction, the completion of the reaction was checked by TLC. Then adding a large amount of ice water until insoluble substances are separated out, and directly using the mixture for the next reaction after suction filtration and drying.
Dissolving collected gray solid (0.5g) in 12.0mL pyridine, adding triethylamine and dialkylamine compounds, stirring at 0 deg.C for 1 hr, detecting by TLC, pouring the reaction solution into ice water until insoluble substances are separated out, vacuum filtering, drying the filter cake, and performing preparative thin layer chromatography (SiO)21/5 MeOH/DCM) to give the title compound.
The specific compound structures, substituents, and reactant selections are shown in table 1 below.
TABLE 1 Structure and substituent selection of Compounds in examples 1-4
The structure detection is carried out on the prepared derivatives 3a-3d respectively, and the specific results are as follows:
compound 3 a: the reaction was determined to be a white solid with a calculated yield of 7.2%; 196.3-198.3 ℃ in m.p.; HR-MS (ESI) determined as C38H65N5NaO8：742.4731([M+Na]742.4722.1H NMR (600MHz, DMSO-d6) delta 9.12(s,1H),8.45(d, J-9.6 Hz,1H),7.42(s,1H),7.08(s,1H),6.84(d, J-15.0 Hz,1H),6.30(d, J-15.0 Hz,1H),5.41(s,1H),5.33(s,1H),5.13(d, J-9 Hz,1H),5.10(d, J-10.5 Hz,1H),5.06(d, J-2.5 Hz,1H),4.65(d, J-17.9 Hz,1H),3.65(d, J-18.0, 1H), 3.62-3.57, 3.3.01 (m-3.3H), 3.01-1H, 3.3.3.3.0, 3.3.3.3H, 3.3.0, 3.3.3.01-1H), 3.3.3.3.3.3.0, 3.3.3.3.3H, 3, 3.3.3.1H, 3, 3.3.15 (m-3.3.3, 3.3.0, 3.3, 3.1H), 1H, 3.3.3, 3, 3.0, 3, 4H) 0.88(d, J ═ 6.8Hz,3H), 0.82-0.76 (m, 6H); it can be seen that the product structure is correct.
Compound 3 b: the reaction was determined to be a white solid with a calculated yield of 11.6%; 192.3-195.8 ℃ in m.p.; HR-MS (ESI) determined as C38H64N5O9：756.4523([M+H](+) and found:756.4510.1H NMR (600MHz, DMSO-d6) δ 8.95(s,1H),8.33(d, J-10.0 Hz,1H),7.49(s,1H),7.15(s,1H),6.85(d, J-14.9 Hz,1H),6.14(d, J-15.0 Hz,1H),5.43(s,1H),5.28(s,1H),5.20(d, J-10.2 Hz,1H), 5.14-5.09 (m,2H),4.51(d, J-18.2 Hz,1H),3.71(d, J-18.2 Hz,1H), 3.67-3.42 (m,7H), 3.22-3.12 (m,1H),2.92(s,3H),2.90 (d, J-18.2 Hz,1H), 3.67-3.42 (m,7H), 3.22-3.12 (m,1H),2.92(s,3H), 6.80 (t, 6.8H), 6.7H, 6H, 1H), 6.7H, 1H, 6H, 1H, 6H, 1H, 6H, 1H, 6H, 1H, 6H, 1H, 6; it can be seen that the product structure is correct.
Compound 3 d: the reaction was determined to be a white solid with a calculated yield of 12.3%; 187.8-189.6 ℃ in m.p.; HR-MS (ESI) measurement of Structure C39H65N5NaO9：770.4680([M+H]770.4669.1H NMR (600MHz, DMSO-d6) delta 8.89(s,1H),8.22(d, J-7.9 Hz,1H),7.42(s,1H),7.10(s,1H),6.86(d, J-14.9 Hz,1H),6.11(d, J-15.0 Hz,1H),5.43(s,1H),5.28(s,1H),5.18(d, J-11.1 Hz,1H), 5.14-5.07 (m,2H),4.71(s,1H),4.51(d, J-18.2 Hz,1H), 3.87-3.81 (m,1H),3.71(d, J-15.8, 1H), 3.67-3.56 (m,2H), 3.2H, 1H), 3.81(m, 2H), 3.7.7.7.7.7 (d, 1H), 1H, 7.9H, 1H, 7.0.9 (d, 1H), 1H), 4H) 0.82-0.75 (m, 6H); it can be seen that the product structure is correct.
Examples of the experiments
1. In vitro activity against Clostridium difficile and
the biological activity of the prepared compounds 3a-3d was tested separately, and the in vitro Minimal Inhibitory Concentration (MIC) against Clostridium difficile in vitro was determined, using Rakicidin B1 compound as control (noted as RB 1).
Selecting target compounds with HPLC purities respectively higher than 95%; the 4 compounds are prepared into 1.28mg/mL mother liquor by DMSO at the moment of use, and are stored at low temperature for later use, and are diluted as required when in use.
Anaerobic bacteria culture medium: brucella hemimelitensis broth (5g/mL), vitamin K1(1g/mL), lysed horse blood (5% v/v).
Preparation of bacterial inoculum: the anaerobic bacteria are inoculated on the anaerobic bacteria growth plate 2-3 days in advance. The bacterial concentration is adjusted to 1-2 multiplied by 10 on the same day of experiment6CFU/ml, then 100. mu.l were transferred to a 96-well round bottom plate and 100. mu.l of drug or the same volume of medium was added as a control.All compounds will be dissolved to 1.28mg/ml in DMSO at the concentrations tested: 128. 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 mug/ml. The antibiotics fidaxomicin (fidaxomicin) and metronidazole (metronidazole) will be used as reference compounds. The 96-well round bottom plate obtained above was placed at 37 ℃ under 85% humidity and cultured under anaerobic conditions for 46-48 hours. The bacterial growth is obviously inhibited (inhibited)>90%) will be defined as the MIC of the compound. The test results are shown in table 2 below.
In vitro anti-clostridium difficile Activity of the compounds of Table 2
aMIC Compound (μ g) inhibits growth of 90% of C.difficile resistant bacteria,bs, sensitive to vancomycin and metronidazole;bm is metronidazole resistance;bv, vancomycin resistant type.
cn.d. not detected.
dPositive control sample, RB1: Rakicidin B1.
Therefore, the Rakicidins carbamate derivative provided by the invention has better in-vitro clostridium difficile resistance activity.
2. Anti-tumor cell A549 and HT-29 activity research
The compounds 3a-3d prepared above were tested for anti-tumor activity, cytotoxicity studies on tumor cells A549 and HT-29 under normoxic conditions, and Rakicidin B1 compound (designated RB1) was used as a control.
Samples of Rakicidin B1 (noted RB1) and target compounds 3a-3d were dissolved in DMSO respectively to a concentration of 1mg/mL and then diluted respectively to final concentrations of 0.75. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, 0.005. mu.g/mL and 0.0025. mu.g/mL.
And (3) culturing under normal oxygen: respectively taking tumor cells A549 and HT-29 in exponential growth period, respectively, and respectively seeding in 96-well plate (cell concentration is 105/mL, 100 ul/well), 5% (v/v) CO2Culturing in an incubator for 24hr to adhere to the wall,100 ul/well of fresh medium with drug was added, 3 duplicate wells were set for each concentration, and a blank control well (medium alone) was set as a negative control, 3 duplicate wells were also set. The culture was continued for 72hr, and the culture was terminated.
The supernatant was discarded and 100. mu.L of freshly prepared 0.5mg/mL MTT serum-free medium was added to each well and incubation continued at 37 ℃ for 4 h. The supernatant was carefully discarded, 200. mu.L of DMSO was added to dissolve the MTT formazon precipitate, and after mixing with a micro ultrasonic oscillator, the optical density at 544nm was measured on a microplate reader.
Tumor cell growth inhibition (%) (OD control-OD experiment)/(OD control-OD blank) × 100%.
The test results are shown in table 3 below and fig. 1.
TABLE 3 cytotoxicity Studies of Compounds on tumor cells A549 and HT-29 under normoxic conditions
aIC50The half growth inhibition rate of the tumor is determined by MTT method, each experiment is repeated three times, and the average value is taken. .
bHT-29 is human intestinal cancer cells; b A549 non-small lung cancer cells.
cPositive control drug, RB1: Rakicidin B1.
3. Research on activity of anti-tumor cells Caco-2
The prepared compound 3B is subjected to tumor cell Caco-2 antitumor activity detection, cytotoxicity research on the tumor cell Caco-2 under normoxic and hypoxic conditions is detected, and a Rakicidin B1 compound (recorded as RB1) is used as a control.
Samples of Rakicidin B1 and target compound 3B were dissolved in DMSO to a concentration of 1mg/mL, respectively, and then diluted to final concentrations of 0.75. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, 0.005. mu.g/mL and 0.0025. mu.g/mL, respectively.
And (3) culturing under normal oxygen: caco-2 cells in exponential growth period were seeded in 96-well plates (cell concentration 105/mL, 100 ul/well) with 5% (v/v) CO2Culturing in incubator for 24hr to allow it to adhere to the wall, adding 100 ul/well fresh culture medium with drug, setting 3 multiple wells per concentration, setting blank control well (adding culture medium only) as negative control, and setting 3 multiple wells similarly. The culture was continued for 72hr, and the culture was terminated.
Hypoxic culture: caco-2 cells in exponential growth phase were seeded in 96-well plates (cell concentration 10)5100 ul/mL) in a hypoxic culture apparatus (94% N) in hypoxic aeration2，5％CO2And 1% of O2V/v)30min, closing the vent valve, then placing the whole device into an incubator at 37 ℃, culturing for 24hr to make the cells adhere to the wall, adding 100 μ l of fresh culture medium with medicine into each hole, setting 3 multiple holes for each concentration, setting a blank control hole (only adding culture medium) as a negative control, and setting 3 multiple holes for the same. Ventilating with oxygen for 30min, closing the ventilation valve, placing into 37 deg.C incubator, culturing for 72hr, and terminating culture.
The supernatant was discarded and 100. mu.L of freshly prepared 0.5mg/mL MTT serum-free medium was added to each well and incubation continued at 37 ℃ for 4 h. The supernatant was carefully discarded, 200. mu.L of DMSO was added to dissolve the MTT formazon precipitate, and after mixing with a micro ultrasonic oscillator, the optical density at 544nm was measured on a microplate reader.
Tumor cell growth inhibition (%) (OD control-OD experiment)/(OD control-OD blank) × 100%.
The results of the tests are shown in Table 4 below and FIG. 2, while the results of the selective studies of the cytotoxicity of Rakicidin B1 and 3B against Caco-2 tumor cells under normoxic and hypoxic conditions are shown in FIG. 3.
TABLE 4 cytotoxicity Studies of Caco-2 tumor cells under normoxic and hypoxic conditions with the compounds Rakicidin B1 and 3B
aIC50The half growth inhibition rate of the tumor is determined by MTT method, each experiment is repeated three times, and the average value is taken.
bCaco-2 cells (1X104cells/well) were seeded in 96-well plates and cultured under normoxic conditions24 hours TheCaco-2 was incubated under normoxic and hypoxic conditions for 72 hours after dosing, and then the median inhibitory concentration was calculated.
From the above results, it can be seen that three of the compounds 3a to 3d synthesized in examples 1 to 4 had a greater or equivalent activity against clostridium difficile than the lead compound; meanwhile, MTT test results showed that the cytotoxicity of the three compounds was reduced, with the highest reduction in cytotoxicity of 3 b. Therefore, the Rakicidin derivative provided by the invention is obtained by modifying and optimizing a Rakicidin B1 mother nucleus structure, and antibacterial and cytotoxic activity screening is carried out on the Rakicidin B1 derivative with a brand-new structure, wherein Rakicidin-3-O- (morphinyl-4-carbonyl formate) retains potential clostridium difficile resistance activity and reduces cytotoxicity most, and the Rakicidin derivative has further potential development value as a clostridium difficile resistance active compound with a brand-new structure type.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof, wherein the derivatives are derivatives obtained by esterification and amination of Rakicidins compounds, and the Rakicidins carbamate derivatives have a structure shown as the following formula (I):
in the formula (I), the compound is shown in the specification,
the R isaAnd RbIndependently of each other, is selected from hydrogen, amino, C1-C10 alkyl, C3-C7 cycloalkyl, C2-C10 alkenyl or C2-C10 alkynyl; alternatively, the first and second electrodes may be,
the R isaAnd RbTogether with the nitrogen atom form a 5-to 10-membered heterocyclic group, other than RaAnd Rb0-4 heteroatoms selected from N, O and S in addition to the nitrogen atom to which it is attached;
the Rakicidins compounds include Rakicidin A, Rakicidin B1, Rakicidin E, Rakicidin G, Rakicidin H or Rakicidin I.
2. Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof according to claim 1, wherein the Rakicidins carbamate derivatives are selected from the group consisting of:
Rakicidin-3-O- (diethyl carbamate);
Rakicidin-3-O- (morphininyl-4-carbonyl formate);
Rakicidin-3-O- (1-methylpiperidine-4-carbamate);
Rakicidin-3-O- (4-hydroxypiperidinyl-1-carboxylate).
3. Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof according to claim 1 or 2, wherein the Rakicidins compound is selected from Rakicidin B1.
4. A process for the preparation of a Rakicidins carbamate derivative as claimed in any one of claims 1 to 3, comprising the steps of:
(1) dissolving a Rakicidins compound with a selected structure in an organic solvent, and adding a chloroformate compound with a selected structure for reaction to obtain an esterification reactant of the Rakicidins compound;
(2) and adding the obtained esterification reactant into organic amine with a selected structure for amination reaction to obtain the Rakicidins carbamate derivative with the required structure.
5. A process for the preparation of Rakicidins carbamate derivatives according to claim 4, wherein:
in the step (1), the reaction temperature is controlled to be-10-0 ℃, and the reaction solvent is a pyridine solvent;
in the step (2), the reaction temperature is controlled to be-5 ℃, and the reaction solvent is a pyridine solvent.
6. Use of the Rakicidins carbamate derivative according to any one of claims 1 to 3, and pharmaceutically acceptable salts, isomers or esters thereof, for the preparation of a medicament against clostridium difficile.
7. Use according to claim 6, wherein the resistance to Clostridium difficile comprises the prevention and/or treatment of diarrhea and/or cancer associated with Clostridium difficile.
8. An anti-clostridium difficile drug, which is characterized in that the Rakicidins carbamate derivative and the pharmaceutically acceptable salt, isomer or ester thereof as claimed in any one of claims 1 to 3 are used as active ingredients, and a pharmaceutically acceptable carrier is added.
9. Use of the Rakicidins carbamate derivative according to any one of claims 1 to 3, and pharmaceutically acceptable salts, isomers or esters thereof for the preparation of a medicament having anti-tumor activity.
10. A medicament having anti-tumor activity, characterized in that the Rakicidins carbamate derivatives and pharmaceutically acceptable salts, isomers or esters thereof as claimed in any one of claims 1 to 3 are used as active ingredients, and a pharmaceutically acceptable carrier is added.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911325504.XA CN111116707B (en) | 2019-12-20 | 2019-12-20 | Rakicidins carbamate derivative and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911325504.XA CN111116707B (en) | 2019-12-20 | 2019-12-20 | Rakicidins carbamate derivative and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111116707A true CN111116707A (en) | 2020-05-08 |
| CN111116707B CN111116707B (en) | 2021-10-22 |
Family
ID=70500606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911325504.XA Active CN111116707B (en) | 2019-12-20 | 2019-12-20 | Rakicidins carbamate derivative and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111116707B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001068121A1 (en) * | 2000-03-17 | 2001-09-20 | Ajinomoto Co., Inc. | Remedies for allergic diseases |
| WO2017166812A1 (en) * | 2016-03-28 | 2017-10-05 | 福建省微生物研究所 | Rakicidin compounds and applications thereof in treating diseases caused by pathogenic anaerobes |
| CN109956993A (en) * | 2017-12-26 | 2019-07-02 | 天津尚德药缘科技股份有限公司 | Rakicidin derivative, preparation method and its is preparing the purposes in anticancer drug |
| CN110437313A (en) * | 2019-08-12 | 2019-11-12 | 福建省微生物研究所 | A kind of Rakicidins esterification derivative and the preparation method and application thereof |
-
2019
- 2019-12-20 CN CN201911325504.XA patent/CN111116707B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001068121A1 (en) * | 2000-03-17 | 2001-09-20 | Ajinomoto Co., Inc. | Remedies for allergic diseases |
| WO2017166812A1 (en) * | 2016-03-28 | 2017-10-05 | 福建省微生物研究所 | Rakicidin compounds and applications thereof in treating diseases caused by pathogenic anaerobes |
| CN109956993A (en) * | 2017-12-26 | 2019-07-02 | 天津尚德药缘科技股份有限公司 | Rakicidin derivative, preparation method and its is preparing the purposes in anticancer drug |
| CN110437313A (en) * | 2019-08-12 | 2019-11-12 | 福建省微生物研究所 | A kind of Rakicidins esterification derivative and the preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| FENG SANG等: "Structure−Activity Relationship Study of Rakicidins: Overcoming Chronic Myeloid Leukemia Resistance to Imatinib with 4‑Methylester-Rakicidin A", 《JOURNAL OF MEDICINAL CHEMISTRY》 * |
| JIAN CHEN等: "Syntheses and anti-pancreatic cancer activities of rakicidin A", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 * |
| 林风等: "缩肽类化合物rakicidins体内外抗艰难梭菌活性研究", 《中国抗生素杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111116707B (en) | 2021-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5661100B2 (en) | Rifamycin derivative | |
| JPH03215428A (en) | Bmy-41950 anti-tumorous antibiotic | |
| Kojiri et al. | A NEW ANTITUMOR SUBSTANCE, BE-18591, PRODUCED BY A STREPTOMYCETE I. FERMENTATION, ISOLATION, PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES | |
| JP2012504559A (en) | Epothilone glycoside compounds and their active ingredients and their applications | |
| CN108485987B (en) | Dibenzoxepin compound and preparation method and application thereof | |
| CN101812079B (en) | Piprazine compound containing polysulfide bond and preparation method and application thereof | |
| CN111116707B (en) | Rakicidins carbamate derivative and preparation method and application thereof | |
| CN110407792B (en) | Secalonic acid J compound derived from penicillium oxalicum and preparation method thereof | |
| CN102030753A (en) | Prenylated indole alkaloids and preparation method and application thereof | |
| KR101764349B1 (en) | A novel flavimycin compound having peptide deformylayse inhibition and antibacterial activity | |
| CN109180673B (en) | Berberine derivative with antibacterial activity and preparation method and application thereof | |
| US10036048B1 (en) | Process for obtaining a naphthoquinone derivative from Streptomyces sp | |
| CN107746423B (en) | Ergosta-7, 22-diene-3-ketoxime, preparation method thereof and application thereof in preparation of antibacterial drugs | |
| EP2638047B1 (en) | "substituted 4-beta-acrylamidopodophyllotoxin congeners as antitumour antibiotics and the process for preparation thereof" | |
| EP0245012A1 (en) | Method for the preparation of 14-hydroxy-6-0-methyl-erythromycin A | |
| CN109134417B (en) | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human cervical cancer | |
| CN114573537B (en) | Compound with anti-neurodegenerative change activity, preparation method and application thereof | |
| JPS61286374A (en) | Antimicrobial and antitumoral phenazinecarboxaldehyde and derivative | |
| CN110669103A (en) | Double demethyl actinomycin derivatives and application thereof in preparation of drug-resistant bacteria infection resisting drugs | |
| CN109956951B (en) | Cervidomycin (Cervinomycin) C1, C2, C3 and C4, and its preparation method and application | |
| KR20050109958A (en) | Gm-95-containing antitumor effect potentiator, combined antitumor preparation and antitumor agent | |
| CN110218174B (en) | Compound and preparation method and application thereof | |
| CN115703788B (en) | Cinnamyl streptomycin and application thereof | |
| CN104211780A (en) | Cyclic depsipeptides compound, preparation method and purpose thereof | |
| CN110403929B (en) | Selenolonic acid M derived from penicillium oxalicum and application thereof in inhibiting human cancer cell proliferation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |