CN103304628A - Nosiheptide derivatives and application thereof - Google Patents
Nosiheptide derivatives and application thereof Download PDFInfo
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- CN103304628A CN103304628A CN2013102246891A CN201310224689A CN103304628A CN 103304628 A CN103304628 A CN 103304628A CN 2013102246891 A CN2013102246891 A CN 2013102246891A CN 201310224689 A CN201310224689 A CN 201310224689A CN 103304628 A CN103304628 A CN 103304628A
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Abstract
The invention discloses novel nosiheptide derivatives and particularly relates to nosiheptide derivatives 1, 2, 3 and 4. The compounds have functions of inhibiting bacterial growth or killing bacteria and can be used for preventing and treating diseases caused by various Gram positive or negative bacteria including drug-resistant strain infection.
Description
Technical field
The present invention relates to contain the compound that a class has anti-microbial effect, be specifically related to the novel nosiheptide analog derivative 1,2,3 and 4 of a class, this compounds has the effect of bacteria growing inhibiting or killing bacteria.
Background technology
Along with the repeatedly Resistant strain that the various microbiotic that use are clinically produced constantly increases, badly influenced human health.Produce clinically multidrug resistant bacterial strain mainly common with gram-positive microorganism, faecalis [the Barrett C T that mainly comprises methicillin resistant staphylococcus aureus, streptococcus pneumoniae and vancomycin resistance, et al, Current opinion in biotechnology, 2003,14 (6): 621-626; Bangert S, et al, Pediatric dermatology, 2012,29 (3): 243-248.].Yet the medicine for the treatment of clinically Gram-negative bacteria is relatively less, in case resistant organism occurs, consequence is with hardly imaginable.Carbapenem antibiotic is brand-new β-lactam antibitics, and Gram-negative bacteria and positive bacteria, aerophil and anerobe etc. are all had super wide spectrum, potent anti-microbial effect, usually is considered to the last method of emergency treatment resistance illness.Therefore, in case infect this bacterioid, patient's life will be subject to grave danger.A lot of Resistant strain produce resistance to Multiple Classes of Antibiotics simultaneously, and the treatment cycle that makes is elongated, and therapeutic dose constantly increases, and also more easily produces new stronger Resistant strain, have had a strong impact on the selectivity for the treatment of and the public's health problem.Yet, in the past in 30 years, come into the market or the new antibacterials developed fewer and feweri, the compound of brand new does not almost have, great majority all are that existing structure is transformed, and make it to reach the purpose that can enter clinical treatment.
Nosiheptide (Nosiheptide) is a member of sulphur peptide antibiotics, has very strong anti-microbial activity, the tool report is by producing in Streptomyces aetuosus ATCC25421, Streptomyces sp.ATCC31463, the Streptomyces glaucoghseus NRRL LL-BP189 fermented liquid, it is a kind of novel nonabsorbable animal feedstuff additive, be suitable for suppressing growth [the Benazet F of gram positive bacterium in the enteron aisle, et al., Experientia, 1980,36 (4): 414-416.].The mechanism that nosiheptide suppresses gram-positive microorganism is to be combined rear interference transcription factor and ribosomal combination with ribosomal protein L 11 and 23S rRNA, thus the synthesizing of arrestin matter [J.M.Harms, et al, Mol.Cell2008,30,26-38].European patent 0073329 discloses generation bacterium of nosiheptide and uses thereof, European patent 0133079 and 0207846 and United States Patent (USP) 5093243 and 4584134 separation purifying technique of nosiheptide is disclosed, United States Patent (USP) 4384043 discloses the zymotechnique of nosiheptide.The people such as Liu Wen have also delivered a kind of preparation method and anti-microbial activity [Liu W, et al., J.Am.Chem.Soc., 2010,132 (46): 16324-16326.] thereof of nosiheptide derivative.But the anti-microbial effect effect of these compounds is unsatisfactory.
Summary of the invention
The present invention is directed to the prior art deficiency, disclose the novel nosiheptide derivative of a class, this analog derivative has no report in above-mentioned document, have very strong anti-Gram-negative bacteria and positive bacteria effect.
Technical solution of the present invention is specific as follows:
Nosiheptide analog derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture with following formula structure:
Wherein, R
1Be H or OH, R
2Be H or OH, R
3For H or
And R
1, R
2Can not be OH simultaneously, perhaps work as R
1Be H, R
2During for OH, R
3Can not be H.
The R of above-mentioned nosiheptide analog derivative
1, R
2And R
3Make up as follows:
R
1Be H, R
2Be H, R
3For H or
R
1Be H, R
2Be OH, R
3For
R
1Be OH, R
2Be H, R
3For H or
Concrete, above-mentioned nosiheptide analog derivative is preferably as follows the compound of structure:
Compound 1
Compound 2:
Compound 3:
Compound 4:
The present invention also provides above-mentioned nosiheptide analog derivative or the application in preparing antibacterial or antibacterials of its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture.
The present invention also provides above-mentioned nosiheptide analog derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture as the application of antiseptic-germicide in agricultural, food, hygiene article sector, such as plant antimicrobial, foodstuff additive, washing product antipathogenic composition etc.
In the above-mentioned application, nosiheptide analog derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture can be used for suppressing or killing gram positive bacterium and gram negative bacterium, further can be used for gram positive bacterium and the gram negative bacterium of resistance.
Above-mentioned nosiheptide analog derivative is expressed preparation by recombinant technology, obtains such as genes involved is knocked out, and also can prepare by the method for chemosynthesis.The bacteriostatic test result of above-mentioned nosiheptide analog derivative shows, but nosiheptide analog derivative establishment of the present invention or kill gram positive bacterium and gram negative bacterium, the especially gram positive bacterium of resistance and gram negative bacterium.
Description of drawings
Fig. 1 is the HPLC color atlas of nosiheptide derivative compound 1 of the present invention.
Fig. 2 is the high resolution mass spectrum figure of nosiheptide derivative compound 1 of the present invention.
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of nosiheptide derivative compound 1 of the present invention.
Fig. 4 is the carbon-13 nmr spectra figure of nosiheptide derivative compound 1 of the present invention.
Fig. 5 is the HPLC color atlas of nosiheptide derivative compound 2 of the present invention.
Fig. 6 is the high resolution mass spectrum figure of nosiheptide derivative compound 2 of the present invention.
Fig. 7 is the hydrogen nuclear magnetic resonance spectrogram of nosiheptide derivative compound 2 of the present invention.
Fig. 8 is the carbon-13 nmr spectra figure of nosiheptide derivative compound 2 of the present invention.
Fig. 9 is the HPLC color atlas of nosiheptide derivative compound 3 of the present invention.
Figure 10 is the high resolution mass spectrum figure of nosiheptide derivative compound 3 of the present invention.
Figure 11 is the hydrogen nuclear magnetic resonance spectrogram of nosiheptide derivative compound 3 of the present invention.
Figure 12 is the carbon-13 nmr spectra figure of nosiheptide derivative compound 3 of the present invention.
Figure 13 is the HPLC color atlas of nosiheptide derivative compound 4 of the present invention.
Figure 14 is the high resolution mass spectrum figure of nosiheptide derivative compound 4 of the present invention.
Figure 15 is the hydrogen nuclear magnetic resonance spectrogram of nosiheptide derivative compound 4 of the present invention.
Figure 16 is the carbon-13 nmr spectra figure of nosiheptide derivative compound 4 of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should understand following examples only for explanation the present invention but not for limiting the scope of the invention.
Slant medium described in following examples, genetic manipulation substratum, seed culture medium and fermentative medium formula (W/V) are as follows:
Slant medium: MS substratum.
Genetic manipulation substratum: ISP1 liquid nutrient medium.
Seed culture medium: soybean cake powder 1.0%, glucose 2.0%, Tryptones 2.0%, yeast extract 1.0%, KNO
30.01%, (NH
4)
2SO
40.02%, FeCl
30.001%, NaCl0.2% and CaCO
30.4%, all the other are water.The pH that goes out to begin is 7.0-7.5.
Fermention medium: soybean cake powder 1.0%, Zulkovsky starch 2.0%, Tryptones 1.0%, yeast extract 0.5%, KNO
30.01%, (NH
4)
2SO
40.02%, FeCl
30.001%, NaCl0.2% and CaCO
30.4%, all the other are water.Initial pH is 7.0-7.5.
1. the structure of gene knockout carrier
1.1 the synthetic design of gene fragment
Nosiheptide biological synthesis gene cluster sequence (GenBank:FJ438820) according to GenBank announces, design respectively following 4 kinds of gene orders:
(1) design nosB gene order is from the 4th base to the sequence deletion 1290 bases;
(2) design nosAB gene order is from the 4th base of nosB gene to the sequence deletion the 438th base of nosA gene;
(3) the nosC gene order is from the 22nd base to the sequence deletion 1216 bases;
(4) the nosBC gene order is from the 22nd base of nosC gene to the sequence deletion the 1290th base of nosB gene.
Design respectively following 4 groups of primers according to above-mentioned 4 kinds of gene orders and be used for corresponding gene amplification:
（1）nosB-inLF：5’-GGC
GAATTCGTGATGCCACGCTCCTGCTCCTGCT-3’EcoRI；
nosB-inLR：5’-ATC
TCTAGAGACCCGGCCGCCGTGGAGTTCACC-3’XbaI；
nosB-F：5’-
TCTAGACATGGGGGCCTCCACGCTCAGATCCGCAC-3’XbaI；
nosB-R：5’-
AAGCTTCGGCGCGGAGGAGTTCTTCGTCGAGTACACCC-3’HindIII。
（2）nosA-HALF：5’-AT
GAATTCGACGTCGTACATCTCGCCCTTGGTC-3’EcoRI；
nosA-HALR：5’-
TCTAGACCGGGCGCCGACGCCTTCATCCCC-3’XbaI；
nosB-F：5’-
TCTAGACATGGGGGCCTCCACGCTCAGATCCGCAC-3’XbaI；
nosB-R：5’-
AAGCTTCGGCGCGGAGGAGTTCTTCGTCGAGTACACCC-3’HindIII。
（3）nosC-HALF：5’-
GAATTCGCGGCTGCGGGCCTGCTTGCGGTTCTTG-3’EcoRI
nosC-HALR：5’-
TCTAGAGTGCGGATCTGAGCGTGGAGGCCCCCATGGAC-3’XbaI
nosC-HAR2F：5’-
TCTAGAGGTGCGCGCTTCGATGTCCACGGCAGGGGCTCC-3’XbaI
nosC-HAR2R：5’-
AAGCTTGGCACCCGCGAACCCGCCCACAGCTGGCAC-3’HindIII。
（4）nosB-inLF：5’-GGC
GAATTCGTGATGCCACGCTCCTGCTCCTGCT-3’EcoRI；
nosB-inLR：5’-ATC
TCTAGAGACCCGGCCGCCGTGGAGTTCACC-3’XbaI；
nosC-HAR2F：5’-
TCTAGAGGTGCGCGCTTCGATGTCCACGGCAGGGGCTCC-3’XbaI
nosC-HAR2R：5’-
AAGCTTGGCACCCGCGAACCCGCCCACAGCTGGCAC-3’HindIII。
Annotate: underscore is restriction enzyme site.
1.2 the chromosomal extraction of S.actuosus
S.actuosus is bought in American Type Culture Collecti (bacterial strain ATCC25421), gets the S.actuosus spore inoculating of-70 ℃ of preservations in the ISP1 liquid nutrient medium, and 30 ℃ of 250rpm cultivate 48h.Then, get 2ml bacterium liquid centrifugal 5min under 12000rpm, abandon supernatant, collect thalline.With aseptic washing thalline 1～2 time, vibration, centrifugal 5min abandons supernatant under the 12000rpm, collects mycelium.Mycelium is resuspended to 480 μ l TE25S damping fluids, adds 20 μ l N,O-Diacetylmuramidases (50mg/ml), mixing, 37 ℃ of water-bath 1h.Add 30 μ l10%SDS, 10 μ l Proteinase Ks (10mg/ml), 37 ℃ of every 15min of water-bath 1h(vibrate gently).The NaCl that adds 100 μ l5mol/L, fully mixing adds 65 μ lCTAB/NaCl(10%CTAB, 0.7M NaCl again), mixing is in 65 ℃ of water-bath 30～60min.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), mixing, in 4 ℃, centrifugal 5min under the 12000rpm transfers to supernatant in another arm, repeats this operation 1 time.Add isopyknic chloroform/primary isoamyl alcohol (24:1), mixing, in 4 ℃, centrifugal 5min under the 12000rpm transfers to supernatant in another arm.The Virahol that adds 0.6-1 times of volume, mixing gently, in 4 ℃, centrifugal 5min abandons supernatant under the 12000rpm.The ethanol 1ml of adding 70% washes DNA, and in 4 ℃, centrifugal 5min abandons supernatant under the 12000rpm.Add dehydrated alcohol 1ml and wash DNA, in 4 ℃, centrifugal 5min abandons supernatant under the 12000rpm, is inverted, and makes it fully dried.Add 50 μ l TER(and contain RNase A20 μ g/ml) the damping fluid dissolving DNA, as the template of purpose fragment amplification.
1.3 the acquisition of gene fragment
PCR system: the 25 μ l precious biotech firm in Prime STAR Max Premix(Dalian), upstream primer 1 μ l, downstream primer 1 μ l, template 1 μ l, DMSO2.5 μ l, distilled water 19.5 μ l, cumulative volume 50 μ l.
The PCR condition: 98 ℃ of 10s, 72 ℃ of 30s/kb repeat 30 circulations, 72 ℃ of 5min.
(1) the 4th base of disappearance is to the nosB gene order of the sequence between 1290 bases
The acquisition of nosB gene upstream and downstream homology arm gene fragment: take S.actuosus ATCC25421 karyomit(e) as template, increase with above-mentioned primer nosB-inLF/nosB-inLR and nosB-F/nosB-R respectively, obtain nosB upstream region of gene homology arm nosB-inL and size that size is 2.0kb and be the nosB downstream homology arm nosB-HR of 2.3kb.
(2) the 4th base of disappearance nosB gene is to the nosAB gene order of the sequence between the 438th base of nosA gene
The acquisition of nosAB gene upstream and downstream homology arm gene fragment: take S.actuosus ATCC25421 karyomit(e) as template, increase with above-mentioned primer nosA-HALF/nosA-HALR and nosB-F/nosB-R respectively, obtain nosA upstream region of gene homology arm nosA-inL and size that size is 1.9kb and be the nosB downstream homology arm nosB-HR of 2.3kb.
(3) the 22nd base of disappearance is to the nosC gene order of the sequence between 1216 bases
The acquisition of nosC gene upstream and downstream homology arm gene fragment: take S.actuosus ATCC25421 karyomit(e) as template, increase with above-mentioned primer nosC-HALF/nosC-HALR and nosC-HAR2F/nosC-HAR2R respectively, obtain nosC upstream region of gene homology arm nosC-HL and size that size is 2.5kb and be the nosC downstream homology arm nosC-HR of 2.5kb.
(4) the 22nd base of disappearance nosC gene is to the nosBC gene order of the sequence between the 1290th base of nosB gene
The acquisition of nosBC gene upstream and downstream homology arm gene fragment: take S.actuosus ATCC25421 karyomit(e) as template, increase with above-mentioned primer nosB-inLF/nosB-inLR and nosC-HAR2F/nosC-HAR2R respectively, obtain nosB upstream region of gene homology arm nosB-inL and size that size is 2.0kb and be the nosC downstream homology arm nosC-HR of 2.5kb.
1.4 the structure of recombinant plasmid
NosB gene knockout plasmid construction: the fragment nosB-inL that above-mentioned order-checking is correct and nosB-HR are connected to carrier pKC1139[Bierman behind double digestion, M., et al, Gene, 1992,116 (1): 43-49.], obtain recombinant plasmid pKL1120.
NosAB gene knockout plasmid construction: the fragment nosA-inL that above-mentioned order-checking is correct is connected to carrier pKL1120 behind double digestion, obtain recombinant plasmid pKL1121.
NosC gene knockout plasmid construction: the fragment nosC-HL that above-mentioned order-checking is correct and nosC-HR are connected to carrier pKC1139 behind double digestion, obtain recombinant plasmid pKL1122.
NosBC gene knockout plasmid construction: the fragment nosB-inL that above-mentioned order-checking is correct is connected to the carrier pKL1122 that cut through same enzyme behind double digestion, obtain recombinant plasmid pKL1123.
1.5 the evaluation of recombinant plasmid
The method that the employing enzyme is cut is respectively to the above-mentioned recombinant plasmid pKL1120 that builds, and pKL1121, pKL1122 and pKL1123 verify, enzyme are cut the plasmid order-checking of verifying, can carry out following research after order-checking is correct.
2. the structure of engineering strain and screening
With above-mentioned four kinds of carriers that build respectively electricity forward ET12567(pUZ8002 to) in, this bacterial strain reference preparation [Flett F, et al, FEMS microbiology letters, 1997,155 (2): 223-229.] makes up 4 kinds of genetic engineering bacteriums.Then inoculate intestinal bacteria ET12567 and in the LB substratum, in (25 μ g/ml kantlex, 50 μ g/ml paraxin, 50 μ g/ml apramycins), cultivate 12h for 37 ℃.With the dilution proportion nutrient solution of fresh LB substratum with 1:100, add the identical microbiotic selection factor, 220rpm, 37 ℃ are cultured to OD
600=0.4.The 10ml nutrient solution is gone in the 50ml centrifuge tube, and the centrifugal 10min of 8000rpm abandons supernatant, adds the fresh LB substratum of 10ml, and resuspended, 8000rpm is centrifugal, and 10min removes supernatant, adds the 1ml fresh culture resuspended again.Collect the fresh spore (〉=10 of S.actuosus ATCC25421
8Individual/as ml), to suspend with 500 μ l2 * YT substratum, 10min is processed in 50 ℃ of heat shocks, then is cooled to room temperature.Get 500 μ l intestinal bacteria suspensions and mix with 500 μ l spores, the mixed solution of getting 100 μ l is coated and is contained 10mmol/L MgCl
2The MS flat board on, 30 ℃ cultivate 16h-20h after, use the 1ml sterilized water, (containing 25 μ l25mg/ml nalidixic acids and 25 μ l50mg/ml apramycins) covers dull and stereotyped, 30 ℃ of cultivations obtained conjugal transfer in 5 days.Select conjugal transfer to the MS flat board, (contain 50 μ g/ml Apr and 25 μ g/ml nalidixic acids) checking.Be coated with positive conjugal transfer and contain 50 μ g/ml Apr and 25 μ g/ml nalidixic acids in MS(), cultivate 2d for 37 ℃, under the selective pressure of the general resistance of peace and temperature, force plasmid generation homologous recombination to be incorporated on the karyomit(e).Picking conjugal transfer is lax the cultivation in the 50ml of nonreactive ISP1 substratum, and 30 ℃ of 220rpm cultivate 3-5 generation (2d/ generation).The bacterium liquid of proper concn is coated on the MS flat board, cultivated 5d for 30 ℃, picking list bacterium colony lines on the MS flat board of nonreactive, and xeroxs to the MS flat board that contains 50 μ g/ml apramycins, cultivates 3-5d for 30 ℃.Grow at the nonreactive flat board, and bacterial strain not long on the anti-flat board is being arranged, namely mutant strain or wild-type are recovered mutant strain, and this class inoculation is cultivated to the ISP1 liquid nutrient medium, then carry out the PCR checking.The mutant strain that obtains thus is respectively L1120, L1121, L1122 and L1123, corresponds to respectively the mutant strain that nosB gene, nosC gene, nosAB gene and nosBC gene knockout obtain.
The preparation of embodiment 2 nosiheptide derivatives
1. the cultivation of engineering bacteria and fermentation
From the freeze-drying pipe, be seeded on the MS substratum above-mentioned 4 kinds of mutant strain L1120, L1121, L1122 and L1123 respectively, in 28-30 ℃ cultivate 4 days after, change over to and pack into through 121 ℃ of autoclavings in the triangular flask of the seed culture medium of 30min, place shaking table to cultivate, wherein, rotating speed is 220rpm, and temperature is 28-30 ℃, and incubation time is 48-50h.Then, seed liquor is inoculated in the triangular flask of sterilized fermention medium, the transferred species amount is 10%, carries out shake flask fermentation and cultivates, and rotating speed is 220rpm, and temperature is 28-30 ℃, and incubation time is 120h.
After seed culture, also can directly change in the fermentor tank of the sterilized 10L of being equipped with fermention medium.The transferred species amount is 10%, ferments in fermentor tank, and mixing speed is 400-500rpm, and temperature is controlled at 28-30 ℃, and air flow is 7-9L/min, cultivates and finishes fermentation in 4-6 days.
2. the separation and purification of expression product
With above-mentioned four kinds of mutant strain L1120, L1121, L1122 and L1123 fermented liquid after fermentation, the ratio according to volume ratio 1:1 adds ethyl acetate extraction 4-8h respectively, and mixing speed is 200-800rpm/min.Abandon lower floor, the supernatant acetic acid ethyl acetate extract carries out vacuum rotary steam, and then the ratio according to 1:2 adds ethyl acetate and normal hexane, and-20 ℃ are spent the night, and the crystal of separating out is crude product.Crude product namely obtains sterling through 3 recrystallizations.
The sterling that above-mentioned four kinds of mutant strain L1120, L1121, L1122 and L1123 fermented liquid are prepared behind recrystallization repeatedly goes up respectively analysis mode liquid phase Shimadzu SCL-2010A and analyzes, and condition is as follows:
Analyze and use post: Waters Symmetry, 150mm * 4.6mm, 5 μ m
Moving phase: A: contain 0.1%TFA in the distilled water
B: contain 0.1%TFA in the acetonitrile
Gradient: gradient elution 0-10min:40%B-55%B; 10-15min:55%B; 15-20min:55%B-60%B; 20-25min:60%B.
Column temperature: 40 ℃
Flow velocity: 1ml/min
Wavelength: 330nm
Sample size: 20 μ l
Color atlas such as Fig. 1, Fig. 5, Fig. 9 and shown in Figure 13, each self-separation obtains a kind of compound altogether from above-mentioned four kinds of mutant bacterias, difference called after compound 1,2,3,4, wherein compound 1 is from bacterial strain L1120(Δ nosB) separate the fermented liquid and obtain, compound 2 is from bacterial strain L1121(Δ nosAB) separate the fermented liquid and obtain, compound 3 is from bacterial strain L1122(Δ nosC) separate the fermented liquid and obtain, compound 4 is from bacterial strain L1123(Δ nosBC) separate the fermented liquid and obtain.
(1) compound 1 structure elucidation
As shown in Figure 1, the appearance time of target compound is about 14.1min, and purity is greater than 95%.
Detect (Fig. 2) through high resolution mass spectrum, the accurate molecular weight of target compound is 1205.1596, and molecular formula is C
51H
43N
13O
11S
6Further through in conjunction with hydrogen spectrum (Fig. 3), carbon spectrum (Fig. 4) and nuclear magnetic data (table 1) it being carried out structural analysis.
The NMR nuclear magnetic data of table 1 compound 1
By resolving, the chemical structure that has obtained compound 1 is as follows:
(2) compound 2 structure elucidations
As shown in Figure 5, the appearance time of target compound is about 13.5min, and purity is greater than 95%.
Detect (Fig. 6) through high resolution mass spectrum, the accurate molecular weight of target compound is 1275.1634, and molecular formula is C
54H
45N
13O
13S
6Further through hydrogen spectrum (Fig. 7), carbon spectrum (Fig. 8) and nuclear magnetic data (table 2) it is carried out structural analysis.
The NMR nuclear magnetic data of table 2 compound 2
By resolving, the chemical structure that has obtained compound 2 is as follows:
(3) compound 3 structure elucidations
As shown in Figure 9, the appearance time of target compound is about 18.5min, and purity is greater than 95%.
Detect (Figure 10) through high resolution mass spectrum, the accurate molecular weight of target compound is 1275.1660, and molecular formula is C
54H
45N
13O
13S
6Further through hydrogen spectrum (Figure 11), carbon spectrum (Figure 12) and nuclear magnetic data (table 3) it is carried out structural analysis.
The NMR nuclear magnetic data of table 3 compound 3
By resolving, the chemical structure that has obtained compound 3 is as follows:
(4) compound 4 structure elucidations
As shown in figure 13, the appearance time of target compound is about 19.8min, and purity is greater than 95%.
Detect (Figure 14) through high resolution mass spectrum, the accurate molecular weight of target compound is 1259.1705, and molecular formula is C
54H
45N
13O
12S
6Further through hydrogen spectrum (Figure 15), carbon spectrum (Figure 16) and nuclear magnetic data (table 4) it is carried out structural analysis.
The NMR nuclear magnetic data of table 4 compound 4
By resolving, the chemical structure that has obtained compound 4 is as follows:
The bacteriostatic action of embodiment 3 compound 1-4
For the anti-microbial activity of test compounds being estimated and being compared, adopt coubling dilution that the minimum inhibitory concentration (MIC) of different test strain is detected.In the Mueller-Hinton broth culture, cultivate after 18-24 hour, observe and record the growing state of bacterium, and then obtain minimum inhibitory concentration (μ g/ml) for 37 ℃.
Testing compound to the MIC value of different bacterial strains as shown in the table (unit: μ g/ml), according to the table in numerical value as can be known, following 4 kinds of compounds have stronger bacteriostatic action.
The Analysis of Antimicrobial Activity of table 5 nosiheptide derivative
| | Compound | 1 | |
|
|
| Sarcina lutea ATCC9341 | 0.0016 | 0.0032 | 0.0008 | 0.0008 | |
| Lemon yellow coccus ATCC8411 | 0.0032 | 0.0064 | 0.0016 | 0.0016 | |
| Streptococcus pneumoniae A25781 | 0.08 | 0.16 | 0.08 | 0.08 | |
| Streptococcus pneumoniae/penicillin resistant A12782 | 0.001 | 0.002 | 0.008 | 0.008 | |
| Streptococcus aureus/methicillin-resistance A13452 | 0.08 | 0.3 | 0.16 | 0.1 | |
| Streptococcus aureus/anti-different methicillinum A19841 | 0.08 | 0.5 | 0.32 | 0.3 | |
| Staphylococcus epidermidis A109835 | 0.0008 | 0.0016 | 0.0004 | 0.0004 | |
| Staphylococcus haemolyticus A109861 | 0.0004 | 0.0008 | 0.0008 | 0.0016 | |
| Micrococcus A108721 | 0.0008 | 0.0016 | 0.0032 | 0.064 | |
| Vancomycin-resistant enterococcus A27519 | 0.032 | 0.064 | 0.128 | 0.064 | |
| Streptococcus aureus/methicillin-resistant A9537 | 0.0008 | 0.0016 | 0.0032 | 0.0016 | |
| Streptococcus aureus ATCC29213 | 0.0004 | 0.0008 | 0.0032 | 0.0008 |
| Streptococcus aureus ATCC43300 | 0.0002 | 0.0008 | 0.0032 | 0.0004 |
| Subtilis ATCC6633 | 0.0008 | 0.0016 | 0.25 | 0.0016 |
| Enterococcus faecalis ATCC29212 | 0.016 | 0.032 | 0.128 | 0.032 |
| Enterococcus avium A109824 | 0.06 | 0.18 | 0.5 | 0.05 |
| The positive A33210 of morazella catarrhalis/β-penicillinase | 0.08 | 0.16 | 0.16 | 0.08 |
| The positive A10982 of morazella catarrhalis/β-penicillinase | 0.08 | 0.16 | 0.16 | 0.08 |
| Micrococcus catarrhalis ATCC8193 | 0.0016 | 0.0032 | 0.0064 | 0.0016 |
| Pasteurella multocida ATCC43137 | 0.0032 | 0.0064 | 0.0064 | 0.0032 |
Claims (9)
1. the nosiheptide analog derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or the mixture that have the following formula structure:
Wherein, R
1Be H or OH, R
2Be H or OH, R
3For H or
And R
1, R
2Can not be OH simultaneously, perhaps work as R
1Be H, R
2During for OH, R
3Can not be H.
2. described nosiheptide analog derivative as claimed in claim 1 or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture is characterized in that described compound has following structural formula:
3. described nosiheptide analog derivative as claimed in claim 1 or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture is characterized in that described compound has following structural formula:
4. described nosiheptide analog derivative as claimed in claim 1 or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture is characterized in that described compound has following structural formula:
5. described nosiheptide analog derivative as claimed in claim 1 or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture is characterized in that described compound has following structural formula:
6. the application in preparing antibacterial or antibacterials such as one of claim 1-5 described nosiheptide derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture.
Such as one of claim 1-5 described nosiheptide derivative or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture as the application of antiseptic-germicide in agricultural, food, hygiene article sector.
8. such as the application of claim 6 or 7 described nosiheptide derivatives or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture, it is characterized in that described compound can be used for suppressing or killing gram positive bacterium and gram negative bacterium.
9. the application of nosiheptide derivative as claimed in claim 8 or its pharmacy acceptable salt, ester, enantiomer, diastereomer or mixture is characterized in that gram positive bacterium and gram negative bacterium that described bacterium is resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310224689.1A CN103304628B (en) | 2013-06-06 | 2013-06-06 | Nosiheptide derivative and uses thereof |
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| CN201310224689.1A CN103304628B (en) | 2013-06-06 | 2013-06-06 | Nosiheptide derivative and uses thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111840509A (en) * | 2020-07-28 | 2020-10-30 | 首都医科大学附属北京胸科医院 | Application of Nosiheptide in nontuberculous mycobacterial infection |
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| JP2005336088A (en) * | 2004-05-26 | 2005-12-08 | Taisho Pharmaceut Co Ltd | Thiazole-based compound |
| CN101400684A (en) * | 2005-07-13 | 2009-04-01 | 大正制药株式会社 | Thiazole compound |
| CN101586112A (en) * | 2009-06-19 | 2009-11-25 | 中国科学院上海有机化学研究所 | The biological synthesis gene cluster of promise silk seven peptides |
| CN102453077A (en) * | 2010-10-26 | 2012-05-16 | 中国科学院上海有机化学研究所 | Novel fluoronosiheptide and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2005336088A (en) * | 2004-05-26 | 2005-12-08 | Taisho Pharmaceut Co Ltd | Thiazole-based compound |
| CN101400684A (en) * | 2005-07-13 | 2009-04-01 | 大正制药株式会社 | Thiazole compound |
| CN101586112A (en) * | 2009-06-19 | 2009-11-25 | 中国科学院上海有机化学研究所 | The biological synthesis gene cluster of promise silk seven peptides |
| CN102453077A (en) * | 2010-10-26 | 2012-05-16 | 中国科学院上海有机化学研究所 | Novel fluoronosiheptide and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| YI YU 等: "NosA Catalyzing Carboxyl-Terminal Amide Formation in Nosiheptide Maturation via an Enamine Dealkylation on the Serine-Extended Precursor Peptide", 《J.AM.CHEM.SOC.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840509A (en) * | 2020-07-28 | 2020-10-30 | 首都医科大学附属北京胸科医院 | Application of Nosiheptide in nontuberculous mycobacterial infection |
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