CN104188984B - The application in preparation antibacterials of O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone - Google Patents
The application in preparation antibacterials of O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone Download PDFInfo
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Abstract
The present invention relates to organic synthesis and medicinal chemistry art, be specifically related to O (morpholinyl) ethyl derivative of Cleistanone Cleistanone, preparation method and the purposes in preparation antibacterials thereof.The present invention has synthesized O (morpholinyl) ethyl derivative of a new Cleistanone Cleistanone, and discloses its preparation method.Pharmacological experiment shows, O (morpholinyl) ethyl derivative of the Cleistanone Cleistanone of the present invention has antibacterial effect, has the value of exploitation antibacterials.
Description
Technical field
The present invention relates to organic synthesis and medicinal chemistry art, be specifically related to Cleistanone Cleistanone's
O-(morpholinyl) ethyl derivative, preparation method and its usage.
Background technology
The health and lives of the mankind in the diffusion of pathogenic bacterium and the serious threat that strengthens of drug resistance thereof, and antibacterials are
As routine administration be widely used in acquired immune deficiency syndrome (AIDS), organ transplantation and Chronic consumptions (as cancer, diabetes,
Uremia etc.) treatment, although the antimicrobial agent used clinically at present is (such as ketoconazole, amikacin, celebrating
Big mycin, voriconazole, itraconazole, terbinafine, amphotericin, fluconazol etc.) to skin and shallow table
The curative effect of site infection is preferable, but the cumulative toxicity of these antibacterials is relatively strong, usually causes lesions of liver and kidney, disappears
Change road stimulation, dizziness, allergy etc., so the novel antibacterial medicine finding the mechanism of action unique becomes current medicine
One of focus of research and development.
Helicobacter pylori (Helicobacter pylori, Hp) is a kind of Gram-negative spiral bacteria.Research is aobvious
Showing, helicobacter pylori is the primary pathogenic event of acute and chronic gastritis and taste-blindness rate, and may
Relevant with gastric cancer stomach function regulating mucosa-associated lymphoid tissue (MALT) malignant lymphoma morbidity.Recently, the world defends
Hp is classified as I class carcinogen by raw tissue, and it plays a leading role in stomach cancer development.Currently a popular treatment Hp
The scheme infected is to take proton pump inhibitor (PPI) to add two kinds of antibiotic (clarithromycin, A Moxi simultaneously
Woods, tetracycline, metronidazole etc. select two kinds) triple therapy.The main factor affecting triple therapy is considered
It it is the Hp drug resistance to antibacterial；Another serious problems are that proton pump inhibitor can induce dyspepsia, in a large number
In antibacterial then causes digestive tract, the serious of flora destroys.Therefore, efficient, safe anti-Hp activity one is found
Kind new medicine thing becomes an important and urgent task.
The most global morbidity lungy, in increasing trend, is estimated according to World Health Organization (WHO) (WHO), mesh
The population that the front whole world is infected by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) accounts for the world
/ 3rd of population, wherein 5～10% the infected become tuberculosis patient.China occurs activeness every year
Pulmonary tuberculosis patient 1,300,000 example, wherein infectiousness pulmonary tuberculosis about 600,000 example, wherein infectiousness pulmonary tuberculosis about 60
Ten thousand examples, are one of whole world tuberculosis high burden countries.
Come out one after another from antituberculotics, make treatment lungy play epoch-making change.Yet with tuberculosis
The Case management of patient the most not ten sectional specification, irregular chemotherapy, abuse antituberculotics, make drug resistance of tuberculosis
Situation is day by day serious, and the change of drug resistance tends to multi-medicament drug resistance simultaneously, and this gives preventing and treating lungy
Work causes extreme difficulties.Therefore new antituberculotics, the antitubercular agent of the most anti-multidrug resistance are found
Thing is to protection people's health, significant.
From natural product, find compound or lead compound and carry out structural modification and obtain its derivant, thus
The potential drug obtaining high-efficiency low-toxicity has important value.
At present for antibacterial treatment, specific medicament, major part medicine is not had to alleviate antibacterial symptom clinically
There is inevitable toxic and side effects simultaneously, from natural product, find compound or lead compound and tie
Structure is modified and is obtained its derivant, thus the potential drug obtaining high-efficiency low-toxicity has important value.
The compound Cleistanone Cleistanone that the present invention relates to is one and within 2011, delivers (Van Trinh Thi
Thanh et al.,2011.Cleistanone:A Triterpenoid from Cleistanthus indochinensis with
a New Carbon Skeleton.Volume2011,Issue22,Pages4108 4111, August2011)
Compound, we have carried out structural modification to compound Cleistanone Cleistanone, it is thus achieved that one new
O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone, and its antibacterial activity is evaluated,
It has antibacterial activity.
Summary of the invention
The invention discloses O-(morpholinyl) ethyl derivative of a Cleistanone Cleistanone, its structure
For:
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone of the present invention can pass through following side
Prepared by method:
(1) Cleistanone Cleistanone (I) reacts with glycol dibromide and obtains Cleistanone Cleistanone
O-bromoethyl derivant (II)；
(2) O-bromoethyl derivant (II) of Cleistanone Cleistanone and morpholine generation substitution reaction system
Obtain O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone.
The further preparation method of O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone
For:
(1) 440mg compound Cleistanone Cleistanone (I) is dissolved in 10mL benzene, adds in solution
The tetrabutyl ammonium bromide of 0.04g, the glycol dibromide of 3.760g and 50% sodium hydroxide solution of 6mL；
Mixture stirs 24h at 25 degrees Celsius；After 24h, reactant liquor is poured in frozen water, use dichloromethane immediately
It is extracted twice, merges organic phase solution；Then to organic phase solution successively with water and saturated aqueous common salt washing 3
Secondary, then be dried with anhydrous sodium sulfate, last concentrating under reduced pressure is removed solvent and is obtained product crude product；Product crude product silicon
Gel column chromatography eluting, flowing is mutually: petroleum ether/acetone=100:1, v/v, collects yellow and concentrates elution band and get final product
Yellow solid to O-bromoethyl derivant (II) of Cleistanone Cleistanone.
(2) O-bromoethyl derivant (II) of the Cleistanone Cleistanone of 273mg is dissolved in 20mL
In the middle of acetonitrile, it is added thereto to the Anhydrous potassium carbonate of 345mg, the potassium iodide of 84mg and the morpholine of 1742mg,
Mixture is heated to reflux 8h；Reactant liquor is poured in 30mL frozen water after terminating by reaction, uses equivalent dichloromethane
Extract three times, merge organic facies；Organic facies after merging with water and saturated aqueous common salt washing successively, then use nothing
Aqueous sodium persulfate is dried, and concentrating under reduced pressure is removed solvent and obtained product crude product；Product crude product silica gel column chromatography is purified,
Flowing is mutually: petroleum ether/acetone=100:0.5, v/v, collects yellow and concentrates elution band i.e. to obtain Cleistanone
The yellow solid 185.3mg of O-(morpholinyl) ethyl derivative (III) of Cleistanone.
Compound disclosed by the invention can make pharmaceutically acceptable salt or pharmaceutically acceptable carrier.
Pharmacodynamic experiment shows, O-(morpholinyl) ethyl derivative of the Cleistanone Cleistanone of the present invention
There is preferable antibacterial action.The pharmaceutically acceptable salt of the present invention have with its compound as drug effect.
Further, the experiment in vitro of O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone shows,
Chemicals III has a strongest human body fungi activity, therefore the Cleistanone Cleistanone of the present invention
O-(morpholinyl) ethyl derivative is expected to be used for preparing novel anti-human fungi medicine.
Further, the experiment in vitro of O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone shows,
O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone has the strongest anti Helicobacter pylori activity,
Illustrate the diseases such as acute and chronic gastritis closely-related with helicobacter pylori, duodenal ulcer are come
Saying, O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone is the chemical combination of a great exploitation potential
Thing.It can be directly used for treatment and the preparation of related drugs of corresponding disease.
Further, the experiment in vitro of O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone shows,
O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone has the strongest suppression escherichia coli, fluorescence
Pseudomonas, Staphylococcus aureus, Bacillus proteus, the effect of neogenesis cryptococcus, so Cleistanone
O-(morpholinyl) ethyl derivative of Cleistanone as having the compound of antibacterial actions, and can be expected to
Prepare in anti-bacterial drug and be applied.
Further, according to the result of preliminary test, present invention solid medium dilution method determines this chemical combination
Thing is to bacillus calmette-guerin vaccine, mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus (MDR MTB)
The minimal inhibitory concentration of three kinds of tulases, experimental result confirms the O-(morpholinyl) of Cleistanone Cleistanone
Ethyl derivative has the strongest anti-tubercle bacillus and anti-drug resistance tulase activity, can be as treatment tubercle bacillus affection
The lead compound of disease is it can also be used to tubercular drugs is treated in preparation.
The present invention is further detailed explanation by the following examples, but protection scope of the present invention is not had
Any restriction of body embodiment, but be defined in the claims.
Detailed description of the invention
The preparation of embodiment 1 compound Cleistanone Cleistanone
The preparation method of compound Cleistanone Cleistanone (I) is with reference to Crinis Carbonisatus such as Van Trinh Thi Thanh
Document (Van Trinh Thi Thanh et al., the 2011.Cleistanone:A Triterpenoid from of table
Cleistanthus indochinensis with a New Carbon Skeleton.Volume2011,Issue22,
Pages4108 4111, August2011) method.
The synthesis of O-bromoethyl derivant (II) of embodiment 2 Cleistanone Cleistanone
Compound I (440mg, 1.00mmol) is dissolved in 10mL benzene, in solution, adds tetrabutyl phosphonium bromide
Ammonium (TBAB) (0.04g), glycol dibromide (3.760g, 20.00mmol) and 50% hydrogen of 6mL
Sodium hydroxide solution.Mixture stirs 24h at 25 degrees Celsius.After 24h, reactant liquor is poured in frozen water, vertical
I.e. it is extracted twice with dichloromethane, merges organic phase solution.Then to organic phase solution successively with water and saturated food
Saline washs 3 times, then is dried with anhydrous sodium sulfate, and last concentrating under reduced pressure is removed solvent and obtained product crude product.Produce
Thing crude product silica gel column chromatography purification (flowing is mutually: petroleum ether/acetone=100:1, v/v), collects yellow and concentrates
Elution band i.e. obtains the yellow solid (344mg, 63%) of compound II.
1H NMR(500MHz,DMSO-d6) δ 5.04 (s, 1H), 4.82 (s, 1H), 3.94 (d, J=26.5Hz,
1H), 3.87 (d, J=26.5Hz, 2H), 3.57 (s, 2H), 2.40 (d, J=14.0Hz, 1H), 2.39 (d, J=
14.0Hz,1H),2.27(s,1H),2.21(s,1H),2.15(s,1H),1.82(s,1H),1.62(s,2H),1.57
(d, J=3.3Hz, 1H), 1.54 (d, J=3.3Hz, 1H), 1.50 (d, J=1.2Hz, 1H), 1.47 (d, J=1.2
Hz, 1H), 1.39 (d, J=15.3Hz, 2H), 1.34 (d, J=15.3Hz, 1H), 1.26 (dd, J=32.6,13.7
Hz, 4H), 1.13 (d, J=18.0Hz, 2H), 1.05 (s, 6H), 0.98 (s, 1H), 0.88 (s, 12H), 0.78 (s,
3H),0.74(s,1H)。
13C NMR(125MHz,DMSO-d6)δ216.59(s),154.50(s),105.23(s),74.63(s),
69.85(s),59.71(s),52.55(s),51.21(s),47.92(s),44.10(s),42.25(s),41.73(s),
40.64 (s), 40.16 (s), 38.88 (s), 38.65 (s), 37.21 (s), 36.23 (s), 33.34 (d, J=1.1Hz),
32.96(s),29.91(s),27.18(s),26.03(s),24.23(s),23.96(s),20.77(s),18.48(s),
17.98(s),16.93(s)。
HRMS(ESI)m/z[M+H]+calcd for C32H52BrO2:547.3151；found547.3159.
The synthesis of O-(morpholinyl) ethyl derivative (III) of embodiment 3 Cleistanone Cleistanone
Compound II (273mg, 0.5mmol) is dissolved in the middle of 20mL acetonitrile, is added thereto to anhydrous carbon
Acid potassium (345mg, 2.5mmol), potassium iodide (84mg, 0.5mmol) and morpholine (1742mg, 20mmol),
Mixture is heated to reflux 8h.Reactant liquor is poured in 30mL frozen water after terminating by reaction, uses equivalent dichloromethane
Extract three times, merge organic facies.Organic facies after merging with water and saturated aqueous common salt washing successively, then use nothing
Aqueous sodium persulfate is dried, and concentrating under reduced pressure is removed solvent and obtained product crude product.Product crude product silica gel column chromatography purify (stream
Move and be mutually: petroleum ether/acetone=100:0.5, v/v), collect yellow and concentrate elution band i.e. to obtain Cleistanone's
The yellow solid (185.3mg, 67%) of O-(morpholinyl) ethyl derivative.
1H NMR (500MHz, DMSO-d6) δ 5.11 (s, 1H), 4.81 (s, 1H), 4.39 (s, 1H), 3.51 (d, J=
17.7Hz, 6H), 2.59 (s, 2H), 2.47 (s, 4H), 2.32 (t, J=36.5Hz, 2H), 2.24 (s, 1H), 2.20
(s, 1H), 2.11 (s, 1H), 1.79 (d, J=90.0Hz, 3H), 1.59 (d, J=7.0Hz, 2H), 1.40 (d, J=
4.2Hz, 2H), 1.35 (d, J=14.5Hz, 3H), 1.30 1.16 (m, 4H), 1.11 (d, J=6.5Hz, 2H),
1.02 (s, 6H), 0.82 (d, J=8.5Hz, 13H), 0.73 (s, 3H), 0.65 (s, 1H).
13C NMR(125MHz,DMSO-d6)δ216.37(s),154.55(s),105.98(s),73.28(s),69.13
(s),66.17(s),58.23(s),54.02(s),53.01(s),52.77(s),51.83(s),48.12(s),46.23(s),
42.97(s),41.13(s),40.99(s),39.93(s),38.92(s),38.67(s),37.13(s),36.59(s),
33.21(s),32.13(s),29.41(s),27.38(s),26.01(s),24.33(s),23.12(s),20.13(s),
19.13(s),17.25(s),16.16(s).
HRMS(ESI):m/z[M+H]+calcd for C36H60NO3:554.4573；found:554.4579.
O-(morpholinyl) the ethyl derivative antibacterial activity of embodiment 4 Cleistanone
O-(morpholinyl) ethyl derivative (III) the human body fungus of experimental example 1 Cleistanone Cleistanone
Activity
The experiment of human body fungi activity is the method using concentration dilution, measures in triplicate, the disease of test every time
Former bacterium has trichophyton, microsporum canis and trichophyton tonsurans, and bacterial concentration is 105Individual/mL.Cleistanthus sumafranus (Miq) Muell-Arg-C. Saichikii Merr
O-(dimethylamino) ethyl derivative (III) initial concentration of wood ketone Cleistanone is 50.00 μ g/mL (5%
Dimethyl sulfoxide DMSO), gradient dilution to 0.10 μ g/mL, the bacterium solution of equivalent volumes and test sample mixing
Cultivate in 96 orifice plates (ultimate density of formation has: 25.00,12.50,6.25,3.13,1.56,0.78,
0.39,0.20,0.10 and 0.05 μ g/mL), Human Fungi cultivation temperature is respectively 28 DEG C, incubation time
Observing after 24h, if finding, in the disposal hole not having bacterium colony to be formed, that minimum concentration is that sample is minimum antibacterial dense
Degree, i.e. MIC value.This experiment positive control is ketoconazole, and chemicals III human body fungus the results are shown in Table 1.
O-(morpholinyl) ethyl derivative (III) the human body fungus MIC value of table 1 Cleistanone Cleistanone
(μg/mL)
Conclusion: O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone has the strongest resisting
Human Fungi activity, therefore O-(morpholinyl) ethyl derivative (III) of the Cleistanone Cleistanone of the present invention
It is expected to be used for preparing novel anti-human fungi medicine.
O-(morpholinyl) ethyl derivative (III) the anti-pylorus spiral shell of experimental example 2 Cleistanone Cleistanone
Bacillus activity
1) strains tested
Helicobacter pylori (Helicobacter pylori, Hp) reference culture ATCC43504 is purchased from U.S.'s strain and protects
Deposit center (American Type Culture Collection, ATCC).13 strain Hp clinical strains pick up from Nanjing
Drum tower hospital gastroscope room, city accepts gastroscopic patient；In continuous gastroscopy to peptic ulcer, 12
Duodenum 12 ball inflammation or the patient of gastritis verrucosa, be first defined as Hp positive through RUT experiment, then take gastric antrum
Mucosa 1-2 block, is inoculated in after chopping containing 8% horse serum, trimethoprim (trimethropin, TMP)
1.25g/L, polymyxin (polymyxin) B2500U/L, vancomycin (vancomycin) 10mg/L
In Columbia selectivity agar culture medium, in 37 DEG C of (5%O under micro-oxygen environment2, 10%CO2With 85%
N2) cultivate 72 hours.Collecting antibacterial, through smear Gram’s staining, oxidase, catalase and urease are accredited as
After the positive, passing on pure culture, obtained strains is as experimental strain.
2) strain culturing
We use micro-aerobic bag (purchased from Shanghai Medical Univ) to carry out the strain culturing of HP, and it can be by changing
Learn reaction and produce the micro-aerobic environment required for Hp.
3) biological activity determination
Use paper disk method (Microbiological paper method) to measure compound helicobacter pylori is pressed down
Make use, measure minimum inhibitory concentration (the minimal inhibitory of test sample with agar dilution
concentration,MIC)。
I. paper disk method experiment
(A) prepare culture medium by the Columbia culture medium for preparing after high pressure steam sterilization, be cooled to
50-60 DEG C, adding 8% horse serum or Sheep Blood, mixing is poured in the most sterilized culture dish, every ware 7-10ml,
Culture medium thickness is 1.5mm (sterile working).
(B) switching experimental bacteria (being coated with bacterium) takes 10 diluted with microscale sampler8CFU/ml(1OD660=108
CFU/ml) the bacteria suspension 0.1ml of Hp spreads upon suitable culture dish surface equably.It is inverted in 37 DEG C of drying
Taking out after 15min in case, purpose makes agar surface be dried, standby.
(C) patch sample scraps of paper microscale sampler has taken 6 μ l testing sample (mass concentration 2mg/ml) injections
On the round filter paper of sterilizing.With the blank scraps of paper of the aseptic nipper tweezer scraps of paper containing sample and comparison, by sterile working
The scraps of paper are close to containing bacterio-agar surface respectively, at a certain distance patch a piece of paper sheet.Every kind of bacterium is cooked 3 wares, gained
Result seeks its meansigma methods.
(D) each plate is placed in micro-aerobic bag by cultivation, and sealing is opened gas generator, then is placed in 37 DEG C of trainings
Support and case is cultivated 72h.
(E), after surveying antibacterial circle diameter taking-up flat board, the size of antibacterial circle diameter around each scraps of paper is measured respectively.Ginseng
According to the result of matched group, the result of testing sample sensitive experiment can be drawn.In triplicate.
II. agar dilution measures MIC
(A) first the compound of test is prepared by the preparation of Drug plates with dimethyl sulfoxide (DMSO) solution
Become the mother solution of 0.5mg/ml, then dilute with sterilized water, be finally made into 100.0,80.0,60.0,45.0,40.0,
The concentration series of 35.0,30.0,25.0,20.0,15.0,10.0,5.0 and 2.5 μ g/ml, DMSO is being situated between
Concentration in matter is less than 1%.Prepared by 1ml tests compound solution and is incubated in the 8ml of 50 DEG C
Columbia medium, separately adds the horse serum that 1ml is incubated in 50 DEG C and fully mixes, cast in culture dish
Cooling (in culture dish final concentration of the 10.0 of chemicals, 8.0,6.0,4.5,4.0,3.5,3.0,2.5,
2.0,1.5,1.0,0.5 and 0.25 μ g/ml, if there being bacteriostasis, then MIC value is during i.e. these are worthwhile
One).
(B) switching experimental bacteria (being coated with bacterium) draws, with microscale sampler, 1 × 10 diluted8The bacterium of CFU/ml Hp
Suspension 0.1ml spreads upon culture dish surface equably, is inverted in 37 DEG C of drying bakers and takes out after 15min, mesh
The agar surface that makes be dried, standby.
(C) determine that test dish (is contained: 85%N by MIC at micro-aerobic bag2, 10%CO2And 5%O2In),
It is incubated 37 DEG C to cultivate 72 hours, observes Hp growing state, contrast with blank group, with raw entirely without bacterium
Long sample least concentration is minimum inhibitory concentration (minimal inhibitory concentration, MIC) value.Sun
Property comparison for ampicillin (Ampicillin).
Experimental result
Experiment in vitro shows, O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone has
The strongest anti Helicobacter pylori activity, paper disk method shows that its antibacterial circle diameter is 23mm (ATCC43504).
Show that it can completely inhibit 5 random clinical strains and 1 reference culture with agar dilution
(ATCC43504) growth, minimal inhibitory concentration (MIC) is 2.0 μ g/ml.The positive is made with ampicillin
Comparison, its Cmin (MIC) completely inhibiting 6 strain test bacterium is 2.5 μ g/ml.
Conclusion: O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone has stronger pressing down
Helicobacter pylori processed activity, illustrate for acute and chronic gastritis closely-related with helicobacter pylori, stomach,
From the point of view of the diseases such as duodenal ulcer, O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone
It it is the compound of a great exploitation potential.It can be directly used for treatment and the system of related drugs of corresponding disease
Standby.
O-(morpholinyl) ethyl derivative (III) antibacterium of experimental example 3 Cleistanone Cleistanone lives
Property
Antibacterial activity test is the method using concentration dilution, measures in triplicate every time, and pathogen has greatly in test
Enterobacteria, fluorescent pseudomonas, Staphylococcus aureus, Bacillus proteus, neogenesis cryptococcus, bacterial concentration is
105Individual/mL.O-(morpholinyl) ethyl derivative (III) initial concentration of Cleistanone Cleistanone is 50.00
μ g/mL (5% dimethyl sulfoxide DMSO), gradient dilution to 0.10 μ g/mL, the bacterium solution of equivalent volumes and
Test sample be mixed in 96 orifice plates (ultimate density of formation has: 25.00,12.50,6.25,3.13,
1.56,0.78,0.39,0.20,0.10 and 0.05 μ g/mL), antibacterial culturing temperature is respectively 37 DEG C, training
Observing after supporting time 24h, if finding, in the disposal hole not having bacterium colony to be formed, that minimum concentration is that sample is minimum
Antimicrobial concentration, i.e. MIC value.O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone resists
Bacterium the results are shown in Table 2.
O-(morpholinyl) ethyl derivative (III) the antibacterium MIC value (μ g/mL) of table 2 Cleistanone Cleistanone
Conclusion: O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone has the strongest resisting
Bacterial activity, therefore O-(morpholinyl) ethyl derivative (III) of the Cleistanone Cleistanone of the present invention
It is expected to be used for preparing novel anti-bacterial drug.
O-(morpholinyl) ethyl derivative (III) anti-tubercle bacillus of experimental example 4 Cleistanone Cleistanone
Activity
(1) solid medium dilution method measures O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone
Anti-bacillus calmette-guerin vaccine (BCG) absolute concentration
From inclined-plane, scrape BCG cultures, join in 3ml Middlebrook7H9 broth bouillon,
Adding a small amount of bead, screw test tube cap, in vortex oscillator, high vibration grinds, turbid with standard Maxwell ratio
Pipe (MacFarland No.1) ratio is turbid, is i.e. made into bacillus calmette-guerin vaccine (BCG) bacteria suspension of 1mg/ml.
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone is made into highly concentrated with DMSO
The stock solution of degree, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, by closing of having diluted
O-(morpholinyl) ethyl derivative (III) of HUAMU ketone Cleistanone joins 4ml by required dosage
Middlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, cooling
To 50～55 DEG C), mixing, make O-(morpholinyl) ethyl derivative (III) containing Cleistanone Cleistanone,
Concentration is respectively 6.0ug/ml, 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml,
The isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.
Bacillus calmette-guerin vaccine (BCG) the bacteria suspension inoculating loop that concentration is 1mg/ml is dipped ring of numbers, is inoculated in respectively
The culture medium of O-(morpholinyl) ethyl derivative (III) series concentration containing Cleistanone Cleistanone and sky
On white control medium inclined-plane, being placed in 37 DEG C and cultivate 4～8 weeks, observation experiment result, result is as shown in table 3.
In the present embodiment, Middlebrook7H9 broth bouillon used and Middlebrook7H11 agar are cultivated
Base is the conventional culture medium that those skilled in the art carry out when tulase is cultivated, and its formula uses conventional formulation.
(2) solid medium dilution method measures O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone
Killing Mycobacterium Tuberculosis type strain H37Rv strain absolute concentration
From inclined-plane, scrape mycobacterium tuberculosis type strain H37Rv strain culture, join 3ml
In Middlebrook7H9 broth bouillon, add a small amount of bead, screw test tube cap, in vortex oscillator
Upper high vibration grinds, turbid with standard Maxwell opacity tube (MacFarland No.1) ratio, is i.e. made into 1mg/ml
H37Rv strain bacteria suspension.
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone is made into DMSO respectively
The stock solution of high concentration, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, by dilution well
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone join 4ml by required dosage
Middlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, cooling
To 50～55 DEG C), mixing, make O-(morpholinyl) ethyl derivative (III) containing Cleistanone Cleistanone,
Concentration is respectively 6.0ug/ml, 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml,
The isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.
The H37Rv strain bacteria suspension inoculating loop that concentration is 1mg/ml is dipped ring of numbers, is inoculated in containing Cleistanthus sumafranus (Miq) Muell-Arg-C. Saichikii Merr respectively
The culture medium of O-(morpholinyl) ethyl derivative (III) series concentration of wood ketone Cleistanone and blank
In medium slant, being placed in 37 DEG C and cultivate 4～8 weeks, observation experiment result, result is as shown in table 3.
(3) solid medium dilution method measures O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone
Ad tuberculosis is clinically separated the MTB of resistance to ISRE strain absolute concentration
From inclined-plane scrape mycobacterium tuberculosis be clinically separated the MTB of resistance to ISRE strain (resistance to isoniazid, streptomycin,
Rifampicin, ethambutol mycobacterium tuberculosis are clinically separated post) culture, join 3ml Middlebrook7H9
In broth bouillon, adding a small amount of bead, screw test tube cap, in vortex oscillator, high vibration grinds,
Turbid with standard Maxwell opacity tube (MacFarland No.1) ratio, i.e. it is made into the bacteria suspension of 1mg/ml.
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone is made into DMSO respectively
The stock solution of high concentration, by the tween 80 aseptic ultra-pure water dilution stock solution containing 5% to desired concn, by dilution well
O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone join 4ml by required dosage
Middlebrook7H11 agar culture medium (this culture medium 121 DEG C of high pressure steam sterilizations 15 minutes, cooling
To 50～55 DEG C), mixing, make O-(morpholinyl) ethyl derivative (III) containing Cleistanone Cleistanone,
Concentration is respectively 6.0ug/ml, 4.0ug/ml, 3.0ug/ml, 2.0ug/ml, 1.5ug/ml, 1.0ug/ml,
The isocyatic slant medium of 0.75ug/ml, 0.5ug/ml.
The mycobacterium tuberculosis that concentration is 1mg/ml is clinically separated the MTB of resistance to ISRE strain bacteria suspension inoculating loop
Dip ring of numbers, be inoculated in O-(morpholinyl) ethyl derivative (III) containing Cleistanone Cleistanone respectively
In the culture medium of series concentration and blank medium slant, it is placed in 37 DEG C and cultivates 4～8 weeks, observation experiment
As a result, result is as shown in table 3.
Table 3 solid medium dilution method measures O-(morpholinyl) ethyl derivative of Cleistanone Cleistanone
(III) anti-tubercle bacillus absolute concentration result
Conclusion: O-(morpholinyl) ethyl derivative (III) of Cleistanone Cleistanone has the strongest resisting
Tulase and anti-drug resistance tulase activity, can be as the lead compound for the treatment of tubercle bacillus affection disease, it is possible to
For preparing treatment tubercular drugs.
The system of O-(morpholinyl) the ethyl derivative tablet of embodiment 5 Cleistanone involved in the present invention
Standby
Take in the middle of O-(morpholinyl) ethyl derivative or its pharmaceutically acceptable salt of 20 grams of Cleistanone
One, addition prepares the customary adjuvant 180 grams of tablet, and mixing, conventional tablet presses makes 1000.
The system of O-(morpholinyl) the derivatized composite capsule of embodiment 6 Cleistanone involved in the present invention
Standby
Take in the middle of O-(morpholinyl) ethyl derivative or its pharmaceutically acceptable salt of 20 grams of Cleistanone
One, addition prepares the customary adjuvant such as starch 180 grams of capsule, mixing, encapsulated makes 1000.
Claims (3)
1. a Cleistanone Cleistanone with structure shown in formula III O-(morpholinyl) ethyl derivative and
Pharmaceutically acceptable salt application in preparation antibacterials, is characterized by: described bacterium is tulase,
2. a Cleistanone Cleistanone with structure shown in formula III O-(morpholinyl) ethyl derivative and
Pharmaceutically acceptable salt application in preparation antibacterials, is characterized by: described bacterium is escherichia coli, glimmering
Light pseudomonas, Staphylococcus aureus, Bacillus proteus or neogenesis cryptococcus,
The O-of a kind of Cleistanone Cleistanone with structure shown in formula III (
Quinoline base) application in preparation antibacterials of ethyl derivative and pharmaceutically acceptable salt thereof, it is characterized by:
Described tulase is bacillus calmette-guerin vaccine, mycobacterium tuberculosis type strain H37Rv strain and substance of medicines-resistant branched tubercle bacillus.
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