CN109912577B - Compound for inhibiting tumor related to EB virus and preparation method and application thereof - Google Patents
Compound for inhibiting tumor related to EB virus and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a compound for inhibiting tumors related to EB virus, a preparation method and application thereof, and particularly discloses a compound for inhibiting tumors related to EB virus and other diseases and pharmaceutically acceptable salts thereof, wherein the compound has a structure shown in a formula I. Also discloses application of the compound shown in the formula I or pharmaceutically acceptable salt thereof in preparing medicines for inhibiting EB virus, tumors mediated by the EB virus and other diseases mediated by the EB virus. The compound of the invention can be efficiently and specifically bound on a replication origin of virus DNA, has strong specificity, can interfere the binding of EBNA1 and the replication origin, influences the function of EBNA1 and inhibits the expression of virus genes. And simultaneously, Ht-1 is introduced, so that the capability of polyamide molecules for penetrating a biological membrane and combining target DNA can be enhanced, thereby inhibiting virus replication and leading host cells to die.
Description
Technical Field
The invention relates to the field of medicines, in particular to a compound for inhibiting tumors related to EB virus, a preparation method and application thereof.
Background
The epstein-barr virus is a human herpesvirus, a major oncogenic virus, in addition to causing infectious mononucleosis. In the last 60 th century, it was found to be associated with the African endemic Burkitt lymphoma, and subsequently, EB virus was found to be associated with nasopharyngeal carcinoma, Hodgkin's disease, gastric adenocarcinoma, and the like. The EB virus infects human oral epithelial cells first, followed by B lymphocytes. Some of the virus-bearing B lymphocytes escape the immune response, where the epstein-barr virus enters the latent infection phase. The latent infection type EB virus can express a series of virus proteins, hide surface antigens, change parasitic cells, cause decay and finally cause cancers.
In each latent EB Virus infection, EB Virus Nuclear Antigen type 1 (Epstein-Barr Virus Nuclear Antigen 1, EBNA1) was expressed. Previous studies have shown that EBNA1 is essential for maintaining the survival of viruses and tumor cells. EBNA1 facilitates viral replication and expression of other viral genes by binding to the viral DNA origin of replication. Inhibiting the binding of EBNA1 to the viral origin of replication, such as competitive binding of EBNA1 or the binding site of the origin of replication by small molecules, modification of viral DNA by gene editing, or interference of EBNA1 synthesis by RNA, all have inhibitory effects on viral replication and tumor cell proliferation.
Among many DNA minor groove binders, pyrrole-imidazole polyamide polypeptides have strong DNA sequence recognition specificity. It is composed of pyrrole, imidazole derivative and analogue, can be embedded on DNA minor groove, can efficiently and specifically recognize and combine on target DNA sequence, and can interfere expression of related gene on DNA level.
The Yasuda group of the university of ancient Japan used different synthetic methods to synthesize polyamide molecules against the origin of replication of viral DNA for the inhibition of EB virus (Cancer science.2011,102.12: 2221-. The molecule can recognize 5 base pairs on the virus replication origin, and the semi-inhibitory concentration of the molecule on mouse B95-8 cells containing EB virus reaches 50 mu mol/L. The polyamide molecules synthesized by the method have fewer base pairs and poorer specificity; the semi-inhibitory concentration of the in vitro cell experiment is large and is not enough to be promoted to practical application.
(1) The marketed drugs have no specific drugs aiming at the tumor caused by the EB virus, mainly comprise traditional chemotherapeutic drugs, such as alkylating agents, nucleotide analogues and the like. These drugs have no targeting property and have large side effects.
(2) siRNA and antisense oligonucleotides are difficult to administer because of the difficulty of entering cells. (3) The small molecule drug has obvious off-target effect and correspondingly poor specificity. (4) The prior art which attempts to use polyamide-based polypeptides has the problems of laggard synthesis method, less base recognition and low specificity.
Disclosure of Invention
In order to solve the problems, the invention provides a compound for efficiently and specifically inhibiting tumors and other diseases related to EB virus, which can solve the problems of difficult cell entry and off-target of small-molecule drugs. And because of the characteristics of the existing polyamide molecules, new molecules are synthesized to pair more base pairs, and the specificity of the polyamide molecules is enhanced.
One aspect of the present invention provides a compound for inhibiting EB virus related tumors and other diseases and pharmaceutically acceptable salts thereof, having the structure shown in formula I,
in another aspect, the present invention provides a process for the preparation of a compound of formula I, comprising the steps of:
1) sequentially coupling 1 4-amino-1-methyl-2-carboxy-pyrrole, 1 4-amino-1-methyl-2-carboxy-imidazole and 2 4-amino-1-methyl-2-carboxy-pyrrole on a solid phase resin;
2) coupling carboxyl of 2, 4-diaminobutyric acid on the amino of the pyrrole group at the end of the intermediate obtained in the step 1);
3) sequentially coupling 3 4-amino-1-methyl-2-carboxyl-pyrrole and 1-methyl-2-carboxyl-imidazole on the 4-amino of the 2, 4-diaminobutyric acid of the intermediate obtained in the step 2);
4) and modifying the intermediate obtained in the step 3) with a heuster acid derivative Ht-1, and cracking the resin to obtain a final product.
Wherein, the step 1) is to remove the protective group on the solid-phase resin phenylhydrazine, and couple 1 4-amino-1-methyl-2-carboxyl-pyrrole, 1 4-amino-1-methyl-2-carboxyl-imidazole and 2 4-amino-1-methyl-2-carboxyl-pyrrole.
Preferably, the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:
activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-pyrrole, and coupling with amino on aniline solid-phase synthetic resin or intermediate with a removed protecting group; removing an amino protecting group of the tert-butyloxycarbonyl group;
more preferably, the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:
dissolving 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-pyrrole and triphosgene in an organic solvent together, dropwise adding collidine, adding an alkaline agent after the reaction is completed, coupling and mixing the mixture with amino on the protecting group removing intermediate, and reacting completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O (v: v: v ═ 92:5:2.5) mixed solution.
Preferably, the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) is:
activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-imidazole, and coupling with amino on aniline solid-phase synthetic resin or intermediate with a removed protecting group; removing an amino protecting group of the tert-butyloxycarbonyl group;
more preferably, the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) or 3) is:
dissolving 4-tert-butyloxycarbonylamino-1-methyl-2-carboxyl-imidazole and triphosgene in an organic solvent together, dropwise adding collidine, after the reaction is completed, adding an alkaline agent, coupling and mixing with amino on the aniline solid-phase synthetic resin or the intermediate with the protective group removed, and reacting completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O (v: v: v ═ 92:5:2.5) mixed solution.
In the technical scheme of the invention, step 2) is to activate R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid, and the amino on the intermediate obtained in step 1) is coupled; removing the tert-butyloxycarbonyl protecting group;
preferably, the step 2) is to dissolve R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid and triphosgene into an organic solvent together, dropwise add collidine, after the reaction is completed, add an alkaline agent and a condensing agent until the reaction is completed, perform coupling mixing with the amino on the protecting group-removed intermediate, and perform reaction completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O (v: v: v ═ 92:5:2.5) mixed solution.
In the technical scheme of the invention, the step of coupling 1-methyl-2-carboxyl-imidazole in the step 3) comprises the following steps:
activating carboxyl on 1-methyl-2-carboxylic acid-1H-imidazole, and coupling with amino on the intermediate obtained in the previous step of removing the protecting group; the Fmoc protecting group is subsequently removed.
Preferably, 1-methyl-2-carboxylic acid-1H-imidazole and a condensing agent are dissolved in an organic solvent, an alkaline agent is added for reaction till completion, the mixture is added into the intermediate obtained in the previous step, and the condensation reaction is carried out till completion in an inert atmosphere; the Fmoc protecting group was removed under 20% piperidine/DMF.
In the technical scheme of the invention, the step 4) is to activate the compound A with a condensing agent and an alkaline agent, then react with the product obtained in the step 3) to the completion, and react with dimethylaminopropylamine and Cu (OAc)2The reaction is completed and purified to obtain the final product; the compound A is selected from a herceptic acid derivative Ht-1.
The condensing agent used in the present invention is selected from the group consisting of HATU, HOBt, HCTU, DCC, DIC, EDC, HBTU, HOAt, PyBOP, PyAOP, BOP-Cl, SOCl2And oxalyl chloride.
The alkaline agent refers to a compound capable of providing an alkaline environment, and is selected from one or more of DIEA, collidine and triethylamine.
In a further aspect, the invention provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting epstein-barr virus-mediated tumors and epstein-barr virus-mediated other diseases.
In the technical scheme of the invention, the EB virus mediated tumor refers to malignant lymphosarcoma, lymphoma, nasopharyngeal carcinoma, neuroblastoma, gastric cancer, colorectal cancer and thymic lymphatic epithelial cancer which are positive to the EB virus. Other diseases caused by EB virus refer to mononucleosis.
In a further aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
In the present invention, the term "therapeutically effective amount" refers to a dose administered to a subject for therapeutic purposes, which dose is capable of achieving the purpose of treating the corresponding disease.
Advantageous effects
The inhibitor for the relevant tumors and other diseases caused by the EB virus is pyrrole-imidazole polyamide polypeptide, has polyamide tail end and gamma-aminobutyric acid hairpin structure, can be efficiently and specifically bound on a replication origin of virus DNA, has strong specificity, can interfere the binding of EBNA1 and the replication origin, influences the function of EBNA1, and inhibits the expression of virus genes. And simultaneously, Ht-1 is introduced, so that the capability of polyamide molecules for penetrating through a biological membrane and combining with target DNA can be enhanced, and virus replication is inhibited, and host cells are killed.
Drawings
FIG. 1 is a mass spectrum of a compound of formula I.
FIG. 2A is a comparison of the inhibitory effect of compounds of formula I against proliferation of EB-associated tumor cells and non-EB-associated tumor cells.
FIG. 2B is a graph comparing the effect of compounds of formula I on the relative survival of EB-associated tumor cells and non-EB-associated tumor cells.
FIG. 3A is a graph showing the results of the relative amounts of virus at various time points after the effect of the compound of formula I on Raji cells.
FIG. 3B is a graph showing the results of the relative amounts of virus at various time points after the effect of the compound of formula I on Daudi cells.
FIG. 4 is a graph showing the results of compounds of formula I inhibiting the binding of viral DNA to EBNA 1.
FIG. 5 is a graph showing the results of the inhibition of viral expression by compounds of formula I.
FIG. 6A is a graph of the tumor volume results for the inhibition of growth of virus-bearing tumors by compounds of formula I in tumor-bearing mice.
FIG. 6B is a photograph of tumors in tumor-bearing mice that inhibited growth of virus-bearing tumors with compounds of formula I.
Detailed Description
EXPERIMENTAL EXAMPLE 1 preparation of the Compound of formula I
(a) Swelling resin: a10 mL solid phase reactor was charged with 400mg of Fmoc-protected phenylhydrazine resin (0.66mmol/g, 0.264mmol) and 3mL of CH2Cl2Swelling the resin for 30min, removing CH2Cl2And is ready for use;
(b) removing Fmoc protecting groups: adding 3mL of 20% piperidine/DMF solution to the swollen resin from step (a), N2Bubbling and mixing evenly, after 10min, removing the solvent, and then adding 3mL of 20% piperidine/DMF solution, N2Bubbling and uniformly mixing, washing the resin with DMF (4X 3mL) after 10min, and then washing the resin with 3mL of anhydrous DMF for later use;
(c) amino acid condensation: dissolving 4-tert-butyloxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid (254mg, 1.056mmol) and triphosgene (BTC, 128mg, 0.433mmol) in 2mL of anhydrous THF, slowly adding collidine (488. mu.L, 3.696mmol) dropwise into the solution, reacting to generate a large amount of white precipitate immediately, adding for 3min, adding 2mL of DIEA/DMF solution (5%, v/v), completely removing the white precipitate, transferring the reaction solution to the phenylhydrazine resin with the protective groups removed in the step (b), and adding N to obtain N-methyl-1H-pyrrole-2-carboxylic acid2Bubbling and mixing uniformly, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4X 3mL) for later use;
(d) removing the tert-butyloxycarbonyl protecting group: by CH2Cl2(2X 3mL) washing, CH extraction2Cl23.0mL of TFA/phenol/H was added2Removing the tert-butoxycarbonyl protecting group from the condensation product obtained in step (b) with a mixed solution of O (v: v: v ═ 92:5:2.5), removing the solvent after 2min, and adding 3.0mL of TFA/phenol/H2The mixed solution of O (v: v: v ═ 92:5:2.5) was reacted for 20min with CH2Cl2The resin was washed (2X 3mL) with DMF (4X 3mL) and used3mL of anhydrous DMF was used to wash the resin;
adopting the steps (a), (b), (c) and (d) to obtain the peptide shown in the formula (1) loaded on the phenylhydrazine resin;
(e) amino acid condensation: dissolving 4-tert-butyloxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (255mg, 1.056mmol) and triphosgene (128mg, 0.433mmol) in 1mL of anhydrous THF, slowly adding collidine (488. mu.L, 3.696mmol) dropwise to the solution to react and immediately generate a large amount of white precipitate, adding HOAt (144mg, 1.056mmol), adding 2mL of DIEA/DMF solution (5%, v/v), completely removing the white precipitate, transferring the reaction solution to the peptide of formula (3) obtained in step (f), wherein the peptide is supported on phenylhydrazine resin, and N is N, N is N, N is N, N is S, N is S, N is S2Bubbling and uniformly mixing, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4X 3mL) for later use;
(f) removing Boc protecting group in peptide loaded on phenylhydrazine resin by adopting the step (d) to obtain peptide loaded on phenylhydrazine resin shown in the formula (2);
(g) repeating the deprotection and condensation protection steps (c) and (d), wherein the synthesis of the peptide loaded on the phenylhydrazine resin shown in the formula (3) is completed;
(h) condensation of γ -amino acids: dissolving R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid (465mg, 1.056mmol) and triphosgene (128mg, 0.433mmol) in 2mL of anhydrous THF, slowly adding collidine (488. mu.L, 3.696mmol) dropwise to the solution to react and immediately produce a large amount of white precipitate, adding HOAt (144mg, 1.056mmol) for 1min, adding 2mL of DIEADMF solution (5%, v/v), reaction for 5min, white precipitate completely disappeared, and the reaction solution was transferred to the straight-chain peptide (NH) supported on phenylhydrazine resin represented by formula (3)2-Py-Py-Im-Py-phenylhydrazine resin), N2Bubbling and uniformly mixing, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4 x 3mL) to obtain the peptide shown as a formula (4) loaded on the phenylhydrazine resin;
(i) removing the tert-butyloxycarbonyl protecting group: by CH2Cl2(2X 3mL) washing, CH extraction2Cl23.0mL of TFA/phenol/H was added2Removing the tert-butoxycarbonyl protecting group on the product obtained in the step (H) by using a mixed solution of O (v: v: v ═ 92:5:2.5), removing the solvent after 2min, and adding 3.0mL of TFA/phenol/H again2The mixed solution of O (v: v: v ═ 92:5:2.5) was reacted for 20min with CH2Cl2Washing the resin with 2X 3mL of DMF (4X 3mL) and then with 3mL of anhydrous DMF to obtain the peptide shown in formula (5) loaded on the phenylhydrazine resin;
(j) repeating the deprotection and condensation protection steps (c) and (d), wherein the synthesis of the peptide loaded on the phenylhydrazine resin shown in the formula (6) is completed;
(k) condensation of the terminal amino acids: 1-methyl-1H-imidazole-2-carboxylic acid (132mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) were dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added, reaction was carried out for 5min, and the reaction solution was transferred to the peptide of formula (4) obtained in step (H) supported on phenylhydrazine resin, N2Bubbling, mixing, condensation reacting for 2h, draining the reaction solution, washing the resin with DMF (4X 3mL) to obtain the compound represented by formula (7)A peptide supported on a phenylhydrazine resin;
removing the Fmoc protecting group in the peptide loaded on the phenylhydrazine resin shown in the formula (7) by adopting the step (b);
the herceptic acid derivative Ht-1(539mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) were dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added thereto, reaction was carried out for 5min, the reaction solution was transferred to Fmoc-removed peptide represented by formula (7) supported on phenylhydrazine resin, N2Bubbling, uniformly mixing, carrying out condensation reaction for 1h, pumping out reaction liquid, and washing the resin with DMF (4X 3 mL); the resin was removed and 1mL DMF, 200. mu.L dimethylaminopropylamine and 10mg Cu (OAc) were added2The reaction was shaken at room temperature for 12h, the resin filtered off and charged with 20mL CH2Cl2Washing the resin; the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRAfter 18.5min, the product was collected and lyophilized to give a pale yellow solid compound represented by formula (8), hrms (esi) m/z: theoretical calculation C84H96N27O11[M+H]+1658.7777, found 1658.7794.
The structure of the herceptic acid derivative Ht-1 is as follows (see J.AM. CHEM. SOC.2004, 126, 3736-3747):
experimental example 2 inhibitory Effect of the Compound of formula I on tumor cell growth of Epstein-Barr Virus
Raji cells and Daudi cells carrying EB virus in logarithmic growth phase, and Jurkat cells and MOLT-4 cells containing no virus were seeded in 6-well plates at 50 ten thousand cells per well, and cultured in 2ml of complete RPMI-1640 medium containing compounds of formula I at different concentrations, respectively, so that the drug concentration in each well was 0. mu.M or 10. mu.M, respectively. Wells at a concentration of 0 μ M were added with the same concentration of DMSO as a control. Culturing at 37 deg.C in 5% carbon dioxide cell culture box. On the third day, 2ml of complete RPMI-1640 medium containing the same concentration of the compound of formula I was added to each well. Before adding new culture medium on the third day and on the sixth day, the mixed cell suspension is respectively sucked, stained by 0.4% trypan blue staining solution, counted under a microscope, and the number of living cells is calculated. As shown in FIGS. 2A and 2B, the compound of formula I showed a significant inhibitory effect on the growth of Raji cells and Daudi cells containing viruses, and no significant inhibitory effect on Jurkat cells and MOLT-4 cells containing no viruses.
Raji cells, Daudi cells and B95-8 cells carrying EB virus in logarithmic growth phase, as well as Jurkat cells, MOLT-4 cells, RPMI-8226 cells, Nalm-6 cells and MDA-MB-231 cells without virus were taken. Each well was inoculated in 96-well plates in the appropriate amount (4000 MDA-MB-231, 7000 others) and 100. mu.l of the appropriate medium (complete DMEM high-sugar medium for MDA-MB-231, complete RPMI-1640 medium for others). After MDA-MB-231 cells are inoculated, the cells need to be adhered to the wall. Then 100. mu.l of a suitable medium containing the compound of formula I at various concentrations were added to give a compound concentration of 0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M per well, with 4 wells per concentration. Wells at a concentration of 0 μ M were added with the same concentration of DMSO as a control. After incubation for 72 hours at 37 ℃ in a 5% carbon dioxide incubator, 20. mu.l of 10% MTT in PBS was added to each well, and the mixture was incubated for 30 minutes at 37 ℃ in a 5% carbon dioxide incubator. For Raji et al suspension cells, 96-well plates were centrifuged in a large centrifuge at 1000rpm for 5 minutes. After the medium was discarded, 100. mu.l of DMSO was added to dissolve formazan produced by the cells, the mixture was shaken on a shaker, and the resulting mixture was dissolved at 37 ℃ for 30 minutes. And detecting the light absorption value at 450nm on a microplate reader, and adjusting zero by taking DMSO as a blank. The results are shown in FIG. 2B, where the compound of formula I has significant inhibitory effect on the growth of virus-containing cells and no significant inhibitory effect on virus-free cells.
EXAMPLE 3 inhibition of viral DNA in tumor cells of Epstein-Barr Virus by Compounds of formula I
Raji cells and Daudi cells carrying EB virus in logarithmic growth phase were inoculated in 6-well plates at a ratio of 50 ten thousand cells per Raji and 100 ten thousand cells per Daudi. The culture was carried out in 2ml of complete RPMI-1640 medium containing the compound of formula I at a drug concentration of 10. mu.M in each well, and 2ml of complete RPMI-1640 medium containing the same concentration of DMSO was used as a control, except that the compound of formula I was not added to 1 well. Culturing at 37 deg.C in 5% carbon dioxide cell culture box. Every 24 hours, gently blow one hole, resuspend the cells, and collect into a centrifuge tube. Blanks were collected at 24h in the same manner. Centrifugation at 500 Xg for 5 minutes, media discarded, and pellet washed with 1ml PBS and centrifuged at 500 Xg for 5 minutes. By using
The results of the total cellular DNA extracted from the MicroElute Genomic DNA Kit, the DNA content measured with a microspectrophotometer, and the DNA diluted to 10 ng/. mu.l, were shown in FIGS. 3A and 3B, using EB virus replication origin as a template and human GAPDH gene as an internal control, and the copy number of the viral DNA detected by real-time fluorescent quantitative PCR. FIGS. 3A and 3B show that the compounds of formula I have an inhibitory effect on viral DNA in virus-containing cells, and that the effect increases with time.
EXAMPLE 4 inhibition of EBNA1 binding by EBvirus DNA by Compounds of formula I at in vivo levels
Raji cells carrying EB virus in logarithmic growth phase were inoculated in 150mm dishes at 800, 1200 and 1200 million per dish and cultured in 20ml of complete RPMI-1640 medium containing the compound of formula I so that the drug concentration in each well was 0. mu.M, 10. mu.M and 10. mu.M, respectively. The same concentration of DMSO was added to the 0 μ M dish as a control. Taking an experimental group dish and a control group dish at 24 hours; at 48 hours, another test dish was taken. The resuspended cells were gently pipetted, collected into a centrifuge tube, centrifuged at 250 Xg for 5 minutes, and the supernatant poured into the corresponding dish. The cells were resuspended in 1ml of supernatant, stained with 0.4% trypan blue stain and counted under a microscope to calculate the number of viable cells.
Each group was prepared by adding 800 ten thousand cells back to the corresponding petri dish, shaking and mixing. Then 540. mu.l of 37% formaldehyde was added, followed by rapid shaking and mixing, followed by immobilization for 10 minutes. After which 2ml of 1.25M glycine was added to stop the fixed cells. Centrifugation was carried out at 500 Xg for 5 minutes at 4 ℃ and the supernatant was discarded, and the pellet was washed twice with 20ml of ice PBS and centrifuged under the same centrifugation conditions and the supernatant discarded. Thereafter using
The Enzymatic chrome IP Kit (Magnetic Beads) is used for carrying out a Chromatin co-immunoprecipitation experiment, EBNA1 protein and the cross-linking of corresponding DNA are co-precipitated by using an EBNA1 antibody (1 mu g), and the reaction is detected whether the reaction is nonspecific by using an equal amount of common mouse IgG1 as a negative control antibody. The DNA product obtained by coprecipitation is compared with the total DNA of 2% Input by using a real-time fluorescent quantitative PCR (polymerase chain reaction) by taking a virus replication origin as a template, and the relative content of the DNA product is analyzed. The results are shown in FIG. 4. Figure 4 can illustrate that at in vivo levels, the compound of formula I inhibits the binding of EBNA1 to its binding site compared to the control group.
EXAMPLE 5 inhibition of tumor cell Virus expression products by Compounds of formula I
Raji cells carrying EB virus in logarithmic growth phase were seeded into 6-well plates at 50 ten thousand cells per well. Cultured in 2ml of complete RPMI-1640 medium containing the compound of formula I, at drug concentrations of 0,1,2,5,10 and 20. mu.M per well, respectively. After 24 hours, gently blow and beat the resuspended cells, transfer them to a centrifuge tube, centrifuge for 5 minutes at 500 Xg, discard the medium, wash the pellet with 1ml PBS, centrifuge for 5 minutes at 500 Xg, and discard the supernatant.
Mu.l of 1% Triton X-100 lysate containing 1% protease inhibitor was added, lysed on ice for 20min, and then disrupted with an ultrasonicator 85W for 10 sec. Centrifuging at 4 deg.C for 20min at 20000 × g, and collecting supernatant as protein extract.
After protein quantification by the BCA method, 20 mu g of total protein is taken in each group, loading buffer is added, after denaturation by boiling water bath for 10 minutes, the mixture is spotted on 8% SDS-PAGE separating gel and 5% SDS-PAGE concentrating gel, protein samples are separated by 80-100V electrophoresis, and membrane transfer is carried out on PVDF membrane for 100V 90 minutes.
The membranes were blocked in 5% skim milk in PBST for 30min at room temperature. EBNA1 antibody, EBNA2 antibody, and GAPDH antibody were administered at a ratio of 1:2000 and 1:5000, respectively, and dissolved in 5% skim milk in PBST. The antibodies were incubated with the primary antibody described above and shaken at low speed overnight at 4 ℃. Wash with PBST for 5 minutes each. HRP secondary antibodies of the corresponding species (1:5000 in 5% skim milk in PBST) were then incubated for 1 hour at room temperature. Wash with PBST for 5 minutes each. Development exposure was then performed with a multifunction imager, and the results are shown in fig. 5. FIG. 5 shows that the compound of formula I inhibits the expression of two viral proteins, EBNA1 and EBNA2, the inhibition efficiency is in positive correlation with the drug concentration, and moreover, the compound does not have a significant effect on the expression of GAPDH protein.
Experimental example 6 inhibition of viral tumor growth by Compounds of formula I in tumor-bearing mice
Raji cells grown in log phase and 4-5 week old Balb/c nu mice were taken. Cells were collected by centrifugation at 250 Xg for 5 minutes, the supernatant was discarded, the pellet was washed with PBS, centrifuged under the same conditions, the supernatant was discarded, and the cells were resuspended in FBS-free RPMI medium containing 1X 10 cells per 100. mu.l of the medium7And (4) cells. Each mouse was injected subcutaneously into the flank at 1X 107And (4) cells. When the tumor volume reaches about 100mm3(volume estimation formula: V. L. times.W)2) At that time, administration is started. Experimental groups each mouse was injected peritumorally with 20nmol of the compound of formula I dissolved in 100 μ l of 5% DMSO/normal saline solution. The control group was injected with 100. mu.l of 5% DMSO/physiological saline solution only. Injections were repeated every 3 days and tumor sizes were measured prior to injection, and the results are shown in fig. 6A. Three days after injection 8, the mice were sacrificed, the tumors were stripped off, and the observations were recorded, the results are shown in fig. 6B. The results show that the compound of the formula I has an inhibiting effect on the growth of the virous tumor on tumor-bearing mice.
Claims (12)
1. A compound for inhibiting EB virus related tumor and other diseases and its pharmaceutically acceptable salt, which has the structure shown in formula I,
2. a process for the preparation of a compound of formula I, comprising the steps of:
1) sequentially coupling 1 4-amino-1-methyl-2-carboxy-pyrrole, 1 4-amino-1-methyl-2-carboxy-imidazole and 2 4-amino-1-methyl-2-carboxy-pyrrole on a solid phase resin;
2) coupling carboxyl of 2, 4-diaminobutyric acid on the amino of the pyrrole group at the end of the intermediate obtained in the step 1);
3) sequentially coupling 3 4-amino-1-methyl-2-carboxyl-pyrrole and 1-methyl-2-carboxyl-imidazole on the 4-amino of the 2, 4-diaminobutyric acid of the intermediate obtained in the step 2);
4) modifying the intermediate obtained in the step 3) with a heuster acid derivative Ht-1, and cracking the resin to obtain a final product;
wherein, step 1) is to remove the protective group on the solid resin phenylhydrazine, couple 1 4-amino-1-methyl-2-carboxyl-pyrrole, 1 4-amino-1-methyl-2-carboxyl-imidazole and 2 4-amino-1-methyl-2-carboxyl-pyrrole;
wherein the compound of formula I is as follows:
the structure of the Hersteracid derivative Ht-1 is as follows
3. The method of claim 2, wherein the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:
activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-pyrrole, and coupling with hydrazino on phenylhydrazine solid-phase synthetic resin with a protective group removed or amino on an intermediate; and removing the amino protecting group of the tert-butyloxycarbonyl group.
4. The method according to claim 3, wherein the step of coupling 4-amino-1-methyl-2-carboxy-pyrrole in step 1) or 3) is:
dissolving 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-pyrrole and triphosgene in an organic solvent together, dropwise adding collidine, adding an alkaline agent after the reaction is completed, coupling and mixing the mixture with amino on the protecting group removing intermediate, and reacting completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O mixed solution.
5. The method of claim 2, wherein the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) comprises:
activating carboxyl on 4-tert-butyloxycarbonylamino-1-methyl-2-carboxylic acid-1H-imidazole, and coupling with hydrazino on phenylhydrazine solid-phase synthetic resin with a protective group removed or amino on an intermediate; and removing the amino protecting group of the tert-butyloxycarbonyl group.
6. The method according to claim 2, wherein the step of coupling 4-amino-1-methyl-2-carboxy-imidazole in step 1) or 3) is:
dissolving 4-tert-butyloxycarbonylamino-1-methyl-2-carboxyl-imidazole and triphosgene in an organic solvent together, dropwise adding collidine, after the reaction is completed, adding an alkaline agent, coupling and mixing with a hydrazino group on a phenyl hydrazine solid-phase synthetic resin with a protective group removed or an amino group on an intermediate, and reacting completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O mixed solution.
7. The preparation method according to claim 2, wherein the step 2) is activating R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid, and coupling with amino on the intermediate obtained in the step 1); and removing the tert-butyloxycarbonyl protecting group.
8. The preparation method according to claim 7, wherein the step 2) is that R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid and triphosgene are jointly dissolved in an organic solvent, collidine is dropwise added, after the reaction is completed, an alkaline agent and a condensing agent are added until the reaction is completed, and the mixture is coupled and mixed with the amino group on the protecting group-removed intermediate and is reacted completely in an inert atmosphere; TFA/phenol/H2And removing the tert-butyloxycarbonyl protecting group from the O mixed solution.
9. The method according to claim 2, wherein the step of coupling 1-methyl-2-carboxy-imidazole in step 3) comprises:
activating carboxyl on 1-methyl-2-carboxylic acid-1H-imidazole, and coupling with amino on the intermediate obtained in the previous step of removing a protecting group; the Fmoc protecting group is subsequently removed.
10. The use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting EB virus, EB virus-mediated tumors, and EB virus-mediated other diseases;
the compounds described by formula I are as follows:
11. the use according to claim 10, wherein the EB virus mediated tumor is malignant lymphosarcoma, lymphoma, nasopharyngeal carcinoma, neuroblastoma, gastric carcinoma, colorectal carcinoma, thymic lymphatic epithelial carcinoma positive to EB virus; other diseases mediated by the EB virus are mononucleosis.
12. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier
The compounds described by formula I are as follows:
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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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| 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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