CN109912577A - For inhibiting the compound and its preparation method and application of Epstein-Barr virus related neoplasms - Google Patents
For inhibiting the compound and its preparation method and application of Epstein-Barr virus related neoplasms Download PDFInfo
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Abstract
The present invention relates to a kind of for inhibiting the compound and its preparation method and application of Epstein-Barr virus related neoplasms, specifically disclose it is a kind of for inhibiting the compound and its pharmaceutically acceptable salt of Epstein-Barr virus related neoplasms and other diseases, with structure shown in Formulas I.Also disclose the purposes of compound shown in Formulas I or its pharmaceutically-acceptable salts in the drug for the other diseases that the tumour and Epstein-Barr virus that preparation inhibits Epstein-Barr virus, Epstein-Barr virus to mediate mediate.The compound of the present invention can efficiently be specifically bound on the replication orgin of viral DNA, and high specificity can interfere with the combination of EBNA1 and replication orgin, influence the function of EBNA1, inhibit the expression of viral gene.Introduce Ht-1 simultaneously, can reinforced polyamide molecule penetrate the ability of biomembrane and the ability of combining target DNA, thus suppressing virus replication, cause host cell dead.
Description
Technical field
The present invention relates to field of medicaments, and in particular to for inhibiting compound and its preparation side of Epstein-Barr virus related neoplasms
Method and purposes.
Background technique
Epstein-Barr virus is a kind of nerpes vinrus hominis, and other than leading to infectious mononucleosis, it is still a kind of
Main oncorna virus.Just find that it is related with African region Burkitt lymthoma when the sixties in last century, later again
Discovery Epstein-Barr virus and nasopharyngeal carcinoma, Huo Jinqi disease, sdenocarcinoma of stomach etc. is related successively.Epstein-Barr virus first infects human oral epithelial cells, later
Bone-marrow-derived lymphocyte is infected again.The viruliferous bone-marrow-derived lymphocyte in part can escape immune response, and Epstein-Barr virus therein enters latent infection
Phase.The Epstein-Barr virus of latent infection type can express a series of virus proteins, and hidden face antigen makes parasitic cell change,
Lead to immortalization, finally causes cancer.
In the infection of various Epstein-Barr virus resting form, expression 1 type of eb nuclear antigen (Epstein-Barr Virus
Nuclear Antigen 1,EBNA1).Forefathers studies have shown that EBNA1 to maintain virus and tumour cell existence have must
The property wanted.EBNA1 is by conjunction with the replication orgin of viral DNA, promoting the duplication of virus and the expression of other viral genes.Resistance
Hinder the combination of EBNA1 and virus origin of replication, such as using the binding site of small molecule competitive binding EBNA1 or replication orgin,
Using gene editing modification virus DNA or utilize the synthesis etc. of RNA interference EBNA1, the duplication to virus and tumour cell
Proliferation has inhibiting effect.
In numerous DNA minor groove bindings, pyrrole-imidazole polyamide polypeptide has the identification of very strong DNA sequence dna special
Property.It is made of the derivative and analogue of pyrroles, imidazoles, can be entrenched on DNA ditch, efficient specific recognition and combination
In target dna sequence, the expression of related gene is interfered on DNA level.
The Yasuda group of Japan Nagoya university uses different synthetic methods, has synthesized and has replicated for viral DNA
The polyamide molecule of point, for inhibiting Epstein-Barr virus (Cancer Science.2011,102.12:2221-2230).The molecule energy
It identifies 5 base-pairs on virus origin of replication, reaches 50 μ for the 503nhibiting concentration of the mouse B95-8 cell containing Epstein-Barr virus
mol/L.The base-pair of the polyamide molecule identification of this method synthesis is less, and specificity is poor；In vitro cell experiment it is half-suppressed
Concentration is big, is not enough to be advanced to practical application.
(1) there has been no lead oncogenic specific drug, predominantly classic chemotherapy medicine for Epstein-Barr virus in the drug listed
Object, such as alkylating agent, nucleotide analog.These drugs do not have targeting, and side effect is larger.
(2) siRNA and antisense oligonucleotides are difficult to implement because born of the same parents' difficulty is entered.(3) small-molecule drug undershooting-effect is bright
Aobvious, correspondingly specificity is also poor.(4) existing to attempt that then there is synthetic methods to fall using the technology of polyamide-based polypeptide
Afterwards, the problem that identification base is few, specificity is low.
Summary of the invention
To solve the above-mentioned problems, the present invention provides it is a kind of it is efficient specificity inhibit Epstein-Barr virus related neoplasms and other
The compound of disease is able to solve the problem of small-molecule drug enters born of the same parents' difficulty and misses the target.And due to being directed to existing polyamides
The characteristics of amine molecule, synthesizes new molecule to match more base-pairs, enhances the specificity of polyamide molecule.
One aspect of the invention provide a kind of compound for inhibiting Epstein-Barr virus related neoplasms and other diseases and its
Pharmaceutically acceptable salt, with structure shown in Formulas I,
Another aspect of the invention provides the preparation method of compound of formula I comprising following steps:
1) 1 4- amino -1- methyl -2- carboxyl-pyrroles, 1 4- amino -1- methyl-are successively coupled on solid-phase resin
2- carboxyl-imidazoles and 2 4- amino -1- methyl -2- carboxyl-pyrroles；
2) carboxyl of 2,4-diamino-butanoic is coupled on the amino of the pyrrole radicals of intermediate end obtained by step 1)；
3) 3 4- amino -1- first are successively coupled on 4 bit aminos of the 2,4-diamino-butanoic of the intermediate obtained by step 2)
Base -2- carboxyl-pyrroles, 1 1- methyl -2- carboxyl-imidazoles；
4) modification Hirst acid derivative Ht-1 and on the intermediate obtained by step 3) is carried out, and cracks resin, is obtained eventually
Product.
Wherein, step 1) is the protecting group removed on solid-phase resin phenylhydrazine, is coupled 1 4- amino -1- methyl -2- carboxyl -
Pyrroles, 1 4- amino -1- methyl -2- carboxyl-imidazoles and 2 4- amino -1- methyl -2- carboxyl-pyrroles.
Preferably, step 1) or 3) middle the step of being coupled 4- amino -1- methyl -2- carboxyl-pyrroles are as follows:
Activate 4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- pyrroles on carboxyl, and with deprotection base
Amino on aniline synthesis in solid state resin or intermediate is coupled；Remove the amino protecting group of tertbutyloxycarbonyl；
It is highly preferred that the step of step 1) or 3) middle coupling 4- amino -1- methyl -2- carboxyl-pyrroles are as follows:
4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- pyrroles and triphosgene co-dissolve in organic solvent,
Trimethylpyridine is added dropwise, after fully reacting, carries out coupling with the amino on deprotection base intermediate after addition alkaline agent and mixes,
And under an inert atmosphere to fully reacting；TFA/ phenol/H2O (v:v:v=92:5:2.5) mixed solution removes tertbutyloxycarbonyl
Protecting group.
Preferably, in step 1) the step of coupling 4- amino -1- methyl -2- carboxyl-imidazoles are as follows:
Activate 4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- imidazoles on carboxyl, and with deprotection base
Amino on aniline synthesis in solid state resin or intermediate is coupled；Remove the amino protecting group of tertbutyloxycarbonyl；
It is highly preferred that the step of step 1) or 3) middle coupling 4- amino -1- methyl -2- carboxyl-imidazoles are as follows:
4- t-butoxycarbonyl amino -1- methyl -2- carboxyl-imidazoles and triphosgene co-dissolve are added dropwise in organic solvent
Trimethylpyridine, after fully reacting, be added alkaline agent after with the ammonia on the aniline synthesis in solid state resin or intermediate of deprotection base
Base carries out coupling mixing, and under an inert atmosphere to fully reacting；TFA/ phenol/H2O (v:v:v=92:5:2.5) mixing is molten
Liquid removes tertbutyloxycarbonyl protecting group.
In the inventive solutions, step 2) is activation R-2- (9-fluorenylmethyloxycarbonyl amino) -4- tertbutyloxycarbonyl
Aminobutyric acid, and the amino coupled on the intermediate obtained by step 1)；Remove tertbutyloxycarbonyl protecting group；
Preferably, step 2) is by R-2- (9-fluorenylmethyloxycarbonyl amino) -4- t-butoxycarbonyl amino butyric acid and triphosgene
Trimethylpyridine is added dropwise in organic solvent in co-dissolve, and after fully reacting, alkaline agent and condensing agent is added to fully reacting, and
It carries out coupling with the amino on deprotection base intermediate to mix, and under an inert atmosphere to fully reacting；TFA/ phenol/H2O
(v:v:v=92:5:2.5) mixed solution removes tertbutyloxycarbonyl protecting group.
In the inventive solutions, in step 3) the step of coupling 1- methyl -2- carboxyl-imidazoles are as follows:
Activate 1- methyl -- the carboxyl on 2- carboxylic acid -1H- imidazoles, and with obtained by the preceding step of deprotection base on intermediate
Amino be coupled；Then removing Fmoc protecting group.
Preferably, 1- methyl -2- carboxylic acid -1H- imidazoles and condensing agent are dissolved in organic solvent, be added alkaline agent react to
Completely, before being added in step gained intermediate, condensation reaction is to complete under an inert atmosphere；With 20% piperidines/DMF condition removing
Fmoc protecting group.
In the inventive solutions, step 4) be compound A is activated with condensing agent and alkaline agent, then with
Step 3) products therefrom is reacted to complete, and with dimethylaminopropylamine and Cu (OAc)2To complete, purifying is finally produced for reaction
Object；The compound A is selected from Hirst acid derivative Ht-1.
Condensing agent used in the present invention be selected from HATU, HOBt, HCTU, DCC, DIC, EDC, HBTU, HOAt, PyBOP,
PyAOP、BOP-Cl、SOCl2, one of oxalyl chloride or multiple combinations.
Alkaline agent of the present invention refers to provide the compound of alkaline environment, is selected from DIEA, trimethylpyridine, three second
One of amine is a variety of.
Another aspect of the present invention provides a kind of compound of the present invention or its pharmaceutically-acceptable salts is being made
Purposes in the drug for the other diseases that the standby tumour for inhibiting Epstein-Barr virus to mediate and Epstein-Barr virus mediate.
In the inventive solutions, the tumour that the Epstein-Barr virus mediates refers to the malignant lymphatic of the Epstein-Barr virus positive
Sarcoma, lymthoma, nasopharyngeal carcinoma, neuroblastoma, gastric cancer, colorectal cancer, thymic lymphoma epithelioma.Caused by Epstein-Barr virus its
He refers to monocytosis,mononucleosis at disease.
Another aspect of the present invention provides a kind of pharmaceutical composition comprising therapeutically effective amount it is of the present invention
Compound or its pharmaceutically-acceptable salts.
In the present invention, " therapeutically effective amount " refers in order to which therapeutic purposes are to subject's applied dose, the agent
Amount can achieve the purpose that the corresponding disease for the treatment of.
Beneficial effect
Related neoplasms caused by Epstein-Barr virus of the invention and other diseases inhibitors are pyrrole-imidazole polyamide polypeptide,
With polyamides amine end and γ-aminobutyric acid hairpin structure, can efficiently specifically bind on the replication orgin of viral DNA,
High specificity can interfere with the combination of EBNA1 and replication orgin, influence the function of EBNA1, inhibit the expression of viral gene.Together
When introduce Ht-1, can reinforced polyamide molecule penetrate the ability of biomembrane and the ability of combining target DNA, to inhibit disease
Poison duplication causes host cell dead.
Detailed description of the invention
Fig. 1 is the mass spectrogram of compound of formula I.
Fig. 2A is the inhibitory effect that compound of formula I is proliferated for EB associated tumor cells and non-EB associated tumor cells
Compare.
Fig. 2 B is that compound of formula I is directed to EB associated tumor cells and non-one shadow of EB associated tumor cells relative survival rate
Loud comparison.
Fig. 3 A is result figure of the Formulas I chemical combination to different time points virus relative amount after the effect of Raji cell.
Fig. 3 B is result figure of the Formulas I chemical combination to different time points virus relative amount after the effect of Daudi cell.
Fig. 4 is the result figure that compound of formula I inhibits viral DNA to combine with EBNA1.
Fig. 5 is the result figure that compound of formula I inhibits viral expression product.
Fig. 6 A is that compound of formula I inhibits the gross tumor volume result figure with viral tumour growth on tumor-bearing mice.
Fig. 6 B is that compound of formula I inhibits the tumour photo with viral tumour growth on tumor-bearing mice.
Specific embodiment
The preparation of 1 compound of formula I of experimental example
(a) the fmoc-protected phenylhydrazine resin of 400mg resin swelling: is added in the solid phase reactor of a 10mL
(0.66mmol/g, 0.264mmol) and 3mL CH2Cl2, by resin swelling 30min, extract CH2Cl2, spare；
(b) it removes Fmoc protecting group: 20% piperidines of 3mL/DMF solution being added in the resin after step (a) swelling, N2
It is bubbled and mixes, after 10min, extract solvent, add 20% piperidines of 3mL/DMF solution, N2It is bubbled and mixes, after 10min, use
DMF (4 × 3mL) washs resin, then washs resin with 3mL anhydrous DMF, spare；
(c) amino acid condensation: by 4- t-butoxycarbonyl amino -1- methyl-1 H- pyrroles -2- carboxylic acid (254mg,
1.056mmol) and triphosgene (BTC, 128mg, 0.433mmol) is dissolved in the anhydrous THF of 2mL, and front three is slowly added dropwise into the solution
Yl pyridines (collidine, 488 μ L, 3.696mmol), reaction generate a large amount of white precipitates immediately, add and react 3min, then plus
Enter 2mL DIEA/DMF solution (5%, v/v), white precipitate completely disappears, which is transferred to step (b) deprotection
In the phenylhydrazine resin of base, N2It being bubbled and mixes, 0.5~1h of condensation reaction extracts reaction solution, washs resin with DMF (4 × 3mL),
It is spare；
(d) it removes tertbutyloxycarbonyl protecting group: using CH2Cl2(2 × 3mL) washing, extracts CH2Cl2, 3.0mL is added
TFA/ phenol/H2O (v:v:v=92:5:2.5) mixed solution removes the tertbutyloxycarbonyl on condensation product obtained by step (b) and protects
Base is protected, extracts solvent after 2min, 3.0mL TFA/ phenol/H is added again2The reaction of O (v:v:v=92:5:2.5) mixed solution
20min uses CH2Cl2(2 × 3mL) and DMF (4 × 3mL) wash resin, then wash resin with 3mL anhydrous DMF, spare；
Using step (a), (b), (c) with (d), the peptide being supported on phenylhydrazine resin shown in formula (1) is obtained；
(e) amino acid condensation: by 4- t-butoxycarbonyl amino -1- methyl-1 H- imidazoles -2- carboxylic acid (255mg,
1.056mmol) and triphosgene (128mg, 0.433mmol) is dissolved in the anhydrous THF of 1mL, and trimethyl pyrrole is slowly added dropwise into the solution
Pyridine (488 μ L, 3.696mmol), reaction generate a large amount of white precipitates immediately, add and react 3min, addition HOAt (144mg,
1.056mmol), 2mL DIEA/DMF solution (5%, v/v) is added, white precipitate completely disappears, which is transferred to
It is supported in the peptide on phenylhydrazine resin shown in formula (3) obtained by step (f), N2It is bubbled and mixes, 0.5~1h of condensation reaction, abstraction
Reaction solution washs resin with DMF (4 × 3mL), spare；
(f) the Boc protecting group being supported in the peptide on phenylhydrazine resin is removed using step (d), is obtained shown in formula (2)
The peptide being supported on phenylhydrazine resin；
(g) it repeats to be deprotected and step (c) and (d) is protected in condensation, wherein until completing to obtain being supported on shown in formula (3)
The synthesis of peptide on phenylhydrazine resin；
(h) condensation of gamma-amino acid: by R-2- (9-fluorenylmethyloxycarbonyl amino) -4- t-butoxycarbonyl amino butyric acid
(465mg, 1.056mmol) and triphosgene (128mg, 0.433mmol) are dissolved in the anhydrous THF of 2mL, are slowly added dropwise into the solution
Trimethylpyridine (488 μ L, 3.696mmol), reaction generate a large amount of white precipitates immediately, add reaction 1min, HOAt is added
(144mg, 1.056mmol) adds 2mL DIEA/DMF solution (5%, v/v), reacts 5min, and white precipitate completely disappears,
The reaction solution is transferred to the linear peptides (NH being supported on phenylhydrazine resin shown in formula (3)2- Py-Py-Im-Py- phenylhydrazine tree
Rouge) in, N2It is bubbled and mixes, 0.5~1h of condensation reaction extracts reaction solution, washs resin with DMF (4 × 3mL), obtain formula (4)
Shown in be supported on peptide on phenylhydrazine resin；
(i) it removes tertbutyloxycarbonyl protecting group: using CH2Cl2(2 × 3mL) washing, extracts CH2Cl2, 3.0mL is added
TFA/ phenol/H2O (v:v:v=92:5:2.5) mixed solution removes the tertbutyloxycarbonyl protection on step (h) products therefrom
Base extracts solvent after 2min, and 3.0mL TFA/ phenol/H is added again2The reaction of O (v:v:v=92:5:2.5) mixed solution
20min uses CH2Cl2(2 × 3mL) and DMF (4 × 3mL) wash resin, then wash resin with 3mL anhydrous DMF, obtain formula (5) institute
The peptide being supported on phenylhydrazine resin shown；
(j) it repeats to be deprotected and step (c) and (d) is protected in condensation, wherein until completing to obtain being supported on shown in formula (6)
The synthesis of peptide on phenylhydrazine resin；
(k) condensation of end amino acid: by 1- methyl-1 H- imidazoles -2- carboxylic acid (132mg, 1.056mmol) and PyBOP
(550mg, 1.056mmol) is dissolved in 3mL anhydrous DMF, is added DIEA (350 μ L, 2.112 mmol), 5min is reacted, by the reaction
Liquid is transferred to shown in the resulting formula of step (h) (4) and is supported in the peptide on phenylhydrazine resin, N2It is bubbled and mixes, condensation reaction
2h extracts reaction solution, washs resin with DMF (4 × 3mL), obtain the peptide being supported on phenylhydrazine resin shown in formula (7)；
Using the Fmoc protecting group being supported on shown in step (b) removing formula (7) in the peptide on phenylhydrazine resin；
Hirst acid derivative Ht-1 (539mg, 1.056mmol) and PyBOP (550mg, 1.056 mmol) are dissolved in 3mL
Anhydrous DMF is added DIEA (350 μ L, 2.112mmol), reacts 5min, which is transferred to formula (7) institute of removing Fmoc
In the peptide being supported on phenylhydrazine resin shown, N2It is bubbled and mixes, condensation reaction 1h extracts reaction solution, washed with DMF (4 × 3mL)
Wash resin；Resin is taken out, 1mL DMF, 200 μ L dimethylaminopropylamines and 10mg Cu (OAc) is added2, rocked at room temperature reaction
12h filters out resin, and with 20mL CH2Cl2Wash resin；Organic phase is concentrated, residue is purified with semi-preparative HPLC:
10% acetonitrile-H2O (TFA containing 1%) Gradient elution 5min, 10% to 100% acetonitrile-H2O (TFA containing 1%) gradient
Elute 25min, retention time TR=18.5min, collects product, and freeze-drying obtains faint yellow solid shown in formula (8)
Close object, HRMS (ESI) m/z: calculated value C84H96N27O11[M+H]+1658.7777 actual measurement: 1658.7794.
The structure of Hirst acid derivative Ht-1 it is following (preparation method referring to J.AM.CHEM.SOC.2004,126,
3736-3747):
Inhibiting effect of 2 compound of formula I of experimental example to the growth of tumour cell with Epstein-Barr virus
The Raji cell and Daudi cell of the carrying Epstein-Barr virus in logarithmic growth phase are taken, and without virus
Jurkat cell and MOLT-4 cell contain various concentration in 2ml respectively by 500,000, every hole cell inoculation in 6 orifice plates
It is cultivated in the complete RPMI-1640 culture medium of compound of formula I, making the drug concentration in every hole is respectively 0 μM or 10 μM.Concentration is 0
μM hole be added same concentrations DMSO as control.37 DEG C are placed in, is cultivated in 5% carbon dioxide cell incubator.Third day
When, then in every hole be added 2ml Formula I compounds containing phase concentration complete RPMI-1640 culture medium.New training is added in third day
It supports before base and at the 6th day, draws the cell suspension of mixing respectively, dyed with 0.4% trypan blue dye liquor, counted under the microscope
Number calculates living cells quantity.As a result as shown in Figure 2 A and 2 B, compound of formula I is to thin containing viral Raji cell and Daudi
The growth of born of the same parents has obvious inhibiting effect, to the Jurkat cell and MOLT-4 cell for being free of virus then without obvious inhibiting effect.
Raji cell, Daudi cell and the B95-8 cell of the carrying Epstein-Barr virus in logarithmic growth phase are taken, and is free of
Jurkat cell, MOLT-4 cell, RPMI-8226 cell, Nalm-6 cell and the MDA-MB-231 cell of virus.Every hole with
(MDA-MB-231 4000, other are that 7000) (MDA-MB-231 is complete DMEM with 100 μ l suitable culture mediums to suitable quantity
High glucose medium, other are complete RPMI-1640 culture medium) it is inoculated in 96 orifice plates.It is needed after MDA-MB-231 cell inoculation
It is adherent to cell.The suitable culture medium of 100 compound of formula I of the μ l containing various concentration is added later, makes every hole compound concentration
0 μM, 5 μM, 10 μM and 20 μM, every concentration sets 4 multiple holes.The DMSO of same concentrations is added as control in the hole that concentration is 0 μM.
After cultivating 72 hours in 37 DEG C, 5% carbon dioxide incubator, the PBS solution of 20 μ l 10%MTT is added in every hole, at 37 DEG C,
It is incubated for 30 minutes in 5% carbon dioxide incubator.To suspension cells such as Raji, by 96 orifice plates in super-magnum centrifuge with
1000rpm is centrifuged 5 minutes.Culture medium is discarded, the first a ceremonial jade-ladle, used in libation that 100 μ l DMSO dissolution cell generates is added, shakes up, sets on shaking table
It is dissolved 30 minutes at 37 DEG C.Light absorption value at 450nm is detected in microplate reader, is returned to zero by blank of DMSO.As a result such as Fig. 2 B institute
Show, compound of formula I has obvious inhibiting effect to the growth containing virocyte, then inhibits to make without obvious to the cell without virus
With.
Inhibition of 3 compound of formula I of experimental example to viral DNA in the tumour cell with Epstein-Barr virus
The Raji cell and Daudi cell for taking the carrying Epstein-Barr virus in logarithmic growth phase, by 500,000, the every hole Raji,
1,000,000, the every hole Daudi cell inoculation is in 6 orifice plates.In 2ml complete RPMI-1640 culture medium containing a compound of formula I
Culture, the drug concentration in every hole is respectively 10 μM, and 1 hole is separately taken to be added without compound of formula I, with the complete RPMI-1640 culture of 2ml
The culture medium of the DMSO containing same concentrations is only added as control in base culture.37 DEG C are placed in, 5% carbon dioxide cell incubator
Middle culture.It every 24 hours, gently blows and punches, cell is resuspended, collect into centrifuge tube.For 24 hours when receive after the same method
Collect blank control.500 × g is centrifuged 5 minutes, discards culture medium, cleans precipitating with 1ml PBS, 500 × g is centrifuged 5 minutes.WithMicroElute Genomic DNA Kit extracts cell total DNA, measures DNA content with micro-spectrophotometer
Afterwards, DNA is diluted to 10ng/ μ l, using Epstein-Barr virus replication orgin as template, using people GAPDH gene as internal reference, with real-time
The copy number of fluorescence quantitative PCR detection viral DNA, as a result as shown in Figure 3A and Figure 3B.Fig. 3 A and Fig. 3 B can illustrate, Formulas I
Closing object has inhibiting effect to the viral DNA in the cell containing virus, and function and effect extend at any time and increase.
The horizontal inhibition to epstein barr virus dna in conjunction with EBNA1 in vivo of 4 compound of formula I of experimental example
The Raji cell for taking the carrying Epstein-Barr virus in logarithmic growth phase, is connect by every ware 8,000,000,12,000,000 and 12,000,000
Kind, to cultivate in 20ml complete RPMI-1640 culture medium containing a compound of formula I, keeps the drug in every hole dense in 150mm ware
Degree is respectively 0 μM, 10 μM and 10 μM.The DMSO of same concentrations is added as control in the ware that concentration is 0 μM.At 24 hours, one is taken
A experimental group ware and control group ware；At 48 hours, another experimental group ware is taken.Gently cell is resuspended in piping and druming, is collected into centrifuge tube
In, 250 × g is centrifuged 5 minutes, and supernatant is poured into corresponding ware.Cell is resuspended with 1ml supernatant, is contaminated with 0.4% trypan blue
It is counted under the microscope after liquid dyeing, calculates living cells quantity.
Every group takes 8,000,000 cells, and in the corresponding culture dish of add-back, oscillation is mixed.540 μ l, 37% formaldehyde is added,
Quick oscillation mixes, and fixes 10 minutes.2ml 1.25M glycine is added later and terminates fixed cell.500 × g centrifugation 5 at 4 DEG C
Minute, supernatant is abandoned, precipitating is cleaned twice with 20ml ice PBS, is centrifuged with same centrifugal condition and abandons supernatant.It uses laterIt is real that Enzymatic Chromatin IP Kit (Magnetic Beads) carries out chromatin immune co-precipitation
It tests, is negative right with the common mouse IgG 1 of equivalent with the crosslinking of EBNA1 antibody (1 μ g) co-precipitation EBNA1 albumen and corresponding DNA
According to antibody, whether detection reaction has non-specificity.Obtained DNA product is co-precipitated using virus origin of replication as template, with
The total DNA of 2%Input is compared, and analyzes its relative amount with real-time fluorescence quantitative PCR.As a result as shown in Figure 4.Fig. 4 it may be said that
Bright, compared with the control group, horizontal in vivo, compound of formula I inhibits the combination in the site in connection EBNA1.
Inhibition of 5 compound of formula I of experimental example to tumour cell viral expression product
The Raji cell for taking the carrying Epstein-Barr virus in logarithmic growth phase, by 500,000, every hole cell inoculation in 6 orifice plates
In.It is cultivated in 2ml complete RPMI-1640 culture medium containing a compound of formula I, the drug concentration in every hole is respectively 0,1,2,
5,10 and 20 μM.It after 24 hours, after gently cell is resuspended in piping and druming, is transferred in centrifuge tube, 500 × g is centrifuged 5 minutes, discards training
Base is supported, cleans precipitating with 1ml PBS, 500 × g is centrifuged 5 minutes, abandons supernatant.
The 1%Triton X-100 lysate that 50 μ l contain 1% protease inhibitors is added, cracks 20min on ice,
It is 10 seconds broken with sonicator 85W later.20000 × g is centrifuged 20 minutes at 4 DEG C, takes supernatant, i.e. protein extract.
After carrying out protein quantification using BCA method, every group takes 20 μ g total proteins, and loading buffer, boiling water bath 10 is added
After minute denaturation, albumen is separated by electrophoresis in the point sample on 8%SDS-PAGE separation gel and 5%SDS-PAGE concentration glue, 80-100V
Sample, in 90 minutes transferring films to pvdf membrane of 100V.
By film in the PBST solution of 5% skim milk, room temperature is closed 30 minutes.EBNA1 antibody is configured with 1:500, with
1:2000 configures EBNA2 antibody, configures GAPDH antibody with 1:5000, is dissolved in the PBST solution of 5% skim milk.With
It states primary antibody and is incubated for antibody, low speed shaking table is stayed overnight at 4 DEG C.It is cleaned with PBST, 5 minutes every time.It is incubated for corresponding kind later
HRP secondary antibody (1:5000 is dissolved in the PBST solution of 5% skim milk) is incubated at room temperature 1 hour.It is cleaned with PBST, 5 points every time
Clock.Development exposure is carried out with Multifunctional imaging instrument later, as a result as shown in Figure 5.Fig. 5 shows that compound of formula I inhibits EBNA1
With the expression of EBNA2 both virus proteins, and efficiency is inhibited to be positively correlated with drug concentration, furthermore not to GAPDH egg
White expression generation significantly affects.
6 compound of formula I of experimental example is on tumor-bearing mice to the inhibition with viral tumour growth
The Balb/c nu mouse of the Raji cell and 4-5 week old that take logarithmic phase to grow.250 × g is centrifuged 5 minutes and collects carefully
Born of the same parents abandon supernatant, clean precipitating with PBS, similarity condition centrifugation abandons supernatant, cell is resuspended with the RPMI culture medium without FBS, often
100 μ l culture mediums contain 1 × 107A cell.Every mouse is subcutaneously injected 1 × 10 in side rib7A cell.Reach about to gross tumor volume
100mm3(volumetric estimate formula: V=L × W2) when, start to be administered.Every mouse tumor week of experimental group injects 20nmol Formulas I chemical combination
Object is dissolved in 100 μ l 5%DMSO/ normal saline solutions.Control group only injects 100 μ l 5%DMSO/ normal saline solutions.
Every duplicate injection in 3 days, and the size of tumour is measured before the injection, as a result see Fig. 6 A.Three days after injecting the 8th time, mouse is put to death,
Tumour is stripped out, as a result Fig. 6 B is shown in record observation.The result shows that compound of formula I is on tumor-bearing mice to the life with viral tumour
Length is inhibited.
Claims (10)
1. it is a kind of for inhibiting the compound and its pharmaceutically acceptable salt of Epstein-Barr virus related neoplasms and other diseases, have
Structure shown in Formulas I,
2. the preparation method of compound of formula I comprising following steps:
1) 1 4- amino -1- methyl -2- carboxyl-pyrroles, 1 4- amino -1- methyl -2- carboxylic are successively coupled on solid-phase resin
Base-imidazoles and 2 4- amino -1- methyl -2- carboxyl-pyrroles；
2) carboxyl of 2,4-diamino-butanoic is coupled on the amino of the pyrrole radicals of intermediate end obtained by step 1)；
3) 3 4- amino -1- methyl-are successively coupled on 4 bit aminos of the 2,4-diamino-butanoic of the intermediate obtained by step 2)
2- carboxyl-pyrroles, 1 1- methyl -2- carboxyl-imidazoles；
4) modification Hirst acid derivative Ht-1 and on the intermediate obtained by step 3) is carried out, and cracks resin, obtains finished product.
Wherein, step 1) be remove solid-phase resin phenylhydrazine on protecting group, be coupled 1 4- amino -1- methyl -2- carboxyl-pyrroles,
1 4- amino -1- methyl -2- carboxyl-imidazoles and 2 4- amino -1- methyl -2- carboxyl-pyrroles.
3. preparation method according to claim 2, wherein step 1) or 3) middle coupling 4- amino -1- methyl -2- carboxyl -
The step of pyrroles are as follows:
The carboxyl on 4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- pyrroles is activated, and solid with the phenylhydrazine of deprotection base
The amino on diazanyl or intermediate on phase synthesis resin is coupled；Remove the amino protecting group of tertbutyloxycarbonyl；
Preferably, step 1) or 3) middle the step of being coupled 4- amino -1- methyl -2- carboxyl-pyrroles are as follows:
4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- pyrroles and triphosgene co-dissolve are added dropwise three in organic solvent
Picoline after fully reacting, carries out coupling with the amino on deprotection base intermediate after addition alkaline agent and mixes, and in inertia
Atmosphere is down toward fully reacting；TFA/ phenol/H2O mixed solution removes tertbutyloxycarbonyl protecting group.
4. preparation method according to claim 2, in step 1) the step of coupling 4- amino -1- methyl -2- carboxyl-imidazoles
Are as follows:
The carboxyl on 4- t-butoxycarbonyl amino -1- methyl -2- carboxylic acid -1H- imidazoles is activated, and solid with the phenylhydrazine of deprotection base
The amino on diazanyl or intermediate on phase synthesis resin is coupled；Remove the amino protecting group of tertbutyloxycarbonyl.
5. preparation method according to claim 2, step 1) or 3) middle coupling 4- amino -1- methyl -2- carboxyl-imidazoles
Step are as follows:
Front three is added dropwise in organic solvent in 4- t-butoxycarbonyl amino -1- methyl -2- carboxyl-imidazoles and triphosgene co-dissolve
Yl pyridines, after fully reacting, after addition alkaline agent and on the diazanyl or intermediate on the phenylhydrazine synthesis in solid state resin of deprotection base
Amino carry out coupling mixing, and under an inert atmosphere to fully reacting；TFA/ phenol/H2O mixed solution removes tertiary butyloxycarbonyl
Base protecting group.
6. preparation method according to claim 2, step 2) is the activation tertiary fourth oxygen of R-2- (9-fluorenylmethyloxycarbonyl amino) -4-
Carbonylamino butyric acid, and the amino coupled on the intermediate obtained by step 1)；Remove tertbutyloxycarbonyl protecting group；
Preferably, step 2) is that R-2- (9-fluorenylmethyloxycarbonyl amino) -4- t-butoxycarbonyl amino butyric acid and triphosgene is common
Be dissolved in organic solvent, trimethylpyridine be added dropwise, after fully reacting, alkaline agent and condensing agent is added to fully reacting, and with it is de-
Except the amino on protecting group intermediate carries out coupling mixing, and under an inert atmosphere to fully reacting；TFA/ phenol/H2O mixing
Solution removal tertbutyloxycarbonyl protecting group.
7. preparation method according to claim 2, in step 3) the step of coupling 1- methyl -2- carboxyl-imidazoles are as follows:
Activate the carboxyl on 1- methyl -2- carboxylic acid -1H- imidazoles, and with the amino obtained by the preceding step of deprotection base on intermediate
It is coupled；Then removing Fmoc protecting group.
8. tumour that compound shown in Formulas I or its pharmaceutically-acceptable salts inhibit Epstein-Barr virus, Epstein-Barr virus to mediate in preparation and
Purposes in the drug for the other diseases that Epstein-Barr virus mediates.
9. purposes according to claim 8, the tumour that Epstein-Barr virus mediates refers to the malignant lymphosarcoma of the Epstein-Barr virus positive, leaching
Bar tumor, nasopharyngeal carcinoma, neuroblastoma, gastric cancer, colorectal cancer, thymic lymphoma epithelioma；Epstein-Barr virus mediate other diseases be
Monocytosis,mononucleosis.
10. a kind of pharmaceutical composition comprising compound described in the Formulas I of therapeutically effective amount or its pharmaceutically-acceptable salts and
Pharmaceutically acceptable carrier.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011115246A1 (en) * | 2010-03-19 | 2011-09-22 | 学校法人慶應義塾 | Ebna1 function inhibitor |
| CN106068275A (en) * | 2014-01-23 | 2016-11-02 | 瑞泽恩制药公司 | People's antibody of anti-PD 1 |
| CN106674209A (en) * | 2016-12-23 | 2017-05-17 | 深圳先进技术研究院 | Programmed death acceptor 1 gene inhibitor and preparation method and application thereof |
| CN106866635A (en) * | 2015-12-10 | 2017-06-20 | 深圳先进技术研究院 | PLK1 inhibitor and preparation method and application |
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2017
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011115246A1 (en) * | 2010-03-19 | 2011-09-22 | 学校法人慶應義塾 | Ebna1 function inhibitor |
| CN106068275A (en) * | 2014-01-23 | 2016-11-02 | 瑞泽恩制药公司 | People's antibody of anti-PD 1 |
| CN106866635A (en) * | 2015-12-10 | 2017-06-20 | 深圳先进技术研究院 | PLK1 inhibitor and preparation method and application |
| CN106674209A (en) * | 2016-12-23 | 2017-05-17 | 深圳先进技术研究院 | Programmed death acceptor 1 gene inhibitor and preparation method and application thereof |
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| AI YASUDA 等: "DNA ligand designed to antagonize EBNA1 represses Epstein–Barr virus-induced immortalization", 《CANCER SCI》 * |
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