CN1687101A - Nucleotide-like antiviral medicine - Google Patents
Nucleotide-like antiviral medicine Download PDFInfo
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- CN1687101A CN1687101A CN 200510064329 CN200510064329A CN1687101A CN 1687101 A CN1687101 A CN 1687101A CN 200510064329 CN200510064329 CN 200510064329 CN 200510064329 A CN200510064329 A CN 200510064329A CN 1687101 A CN1687101 A CN 1687101A
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- liposome
- antiviral compound
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Abstract
The present invention relates to a nucleoside anti-virus medicine. Said invention provides a modification nucleoside with action for resisting virus, it can be used for preparing medicine for resisting viral infection.
Description
Technical field:
The present invention relates to a kind of ucleotides antiviral, be specifically related to a kind of protonated modification nucleotide; The invention still further relates to the medicinal compositions of this compounds, and the application of described Nucleotide in the preparation antiviral.
Background technology:
Virus disease is one of human biggest threat that is faced.The principal feature of such disease is its hyperinfection (as HIV, SARS etc.), the volatility of virus itself, the carinogenicity (as HPV, EBV, HBV etc.) that some is viral.Especially, although virus itself is the most simply organism of a kind of structure, it is a kind of the most rambunctious pathogenic former being proved to be, and this has proposed unprecedented challenge with regard to the research and development of giving antiviral.
Up to the present, only have a few antiviral clinical obtain more widely-used.Be used for the treatment of common cold virus as Amentadine and infect, Acyclovir is used for the simplexvirus diseases associated, and Ganciclvir is used for and the cytomegalovirus diseases associated, and AZT, ddI, d4T etc. are used for the treatment of acquired immune deficiency syndrome (AIDS) etc.But severe side effect has all appearred in these antiviral, as have a strong impact on neural system renal function and immunocyte.Therefore, the antiviral of searching novel type has become the emphasis of this type of medicament research and development.
Nucleotide is human body metabolism's normal product and precursor, and to the transformation of Nucleotide modification property, the modification nucleotide that obtains can be very little to the potentiality toxicity of human body, might be developed as the effective medicine of a class new type of safe.
Summary of the invention:
The purpose of this invention is to provide the novel antiviral compound of a class.
The present invention finds; with the natural nucleotide is basic structure; the chemosynthesis approach of utilize optimizing reaches modification to glycosyl with some substituting group to 5 ', 3 ' terminal protection; resulting modification nucleotide has the highly efficient anti-virus infection activity; can also increase the stability of Nucleotide, and toxicity is low.
Described modification nucleotide can be used for suppressing duplicating of human disease's virus widely, and its inhibition mechanism is:
1. by participating in viral RNA or DNA building-up process, nucleic acid chains is extended stop;
2. enzyme is duplicated in combination, thereby reaches the inhibition to virus;
3. suppress the ribonucleotide reductase of virus induction, cause the unbalance of intracellular nucleic thuja acid storehouse.
Described human disease's virus includes, but are not limited to: human immunodeficiency virus (HIV), human herpes simplex vicus (HSV), human cytomegalic inclusion disease virus (CMV), human hepatitis B virus (HBV), the sick hepatovirus (HCV) of people, common cold virus (influenza).
The basic structure of the Nucleotide of described modification is:
Wherein, X and Y be respectively 5 ' and 3 ' end blocking group, can be identical or different chemical group.These groups are selected from straight chained alkyl (as ethyl, propyl group, butyl), hydroxyalkyl (as hydroxypropyl, hydroxyl butyl), alkoxyalkyl etc.;
Preferred X or Y are normal-butyl (CH
3CH
2CH
2CH
2-), 4-hydroxyl normal-butyl (HO-CH
3CH
2CH
2CH
2-);
Z is selected from H, replacement or unsubstituted pyrimidyl, replacement or unsubstituted purine radicals; Preferred Z is thymine base and H;
A is selected from H, alkyl, alkoxyl group, alkoxyalkyl, aryl, thiazolinyl, alkyl alcohol, phenylol, enol etc.
Below three compounds (its code name is respectively NBC-2, NBC-3 and NBC-4) be preferred compound of the present invention,
| The compound code name | ????X | ????Y | ????Z | ????A |
| ????NBC-2 | ????HO-CH 3CH 2CH 2CH 2- | ????HO-CH 3CH 2CH 2CH 2- | Thymine base | ??-OCH 3 |
| ????NBC-3 | ????CH 3CH 2CH 2CH 2- | ????CH 3CH 2CH 2CH 2- | Thymine base | ??H |
| ????NBC-4 | ????CH 3CH 2CH 2CH 2- | ????CH 3CH 2CH 2CH 2- | H | ??H |
Structure is:
The present invention can also utilize protonated process that modification nucleotide is carried out acidification.Described protonated or acidifying is meant proton or hydrogen ion is added to process on the proton acceptor of phosphoric acid in the modification nucleotide or blocking group.Along with the PH reduction of compound solution, Nucleotide is increased by the degree of protonated modification thereupon.When protonated Nucleotide is dissolved in neutral water (pH value is about 7), because Nucleotide is by protonated, the PH of the solution of generation also can reduce.
Synthetic method is: with the reaction of base and amidate (amidite), then to 3 ' with 5 ' protect with protecting group.This synthetic method is the single stage method process, need not the chromatographic separation intermediate product.Utilize this route of synthesis, output can reach the number gram to several kilograms, and purity is usually greater than 99%.
Above-mentioned modification nucleotide also can be further through the liposome packing, make it increase permeability and have characteristics such as the body-internal-circulation time is long, antivirus action is better;
Described liposome particles magnitude range is between 60nm～110nm, and preferable range is between 80nm～100nm;
The ratio of modification nucleotide and lipid in the described liposome (medicine fat ratio) is 1: 9～1: 3, and preferred medicine fat ratio is 1: 5～1: 3.
Described liposome contains materials such as neutral lipid, cholesterol or polyethylene glycols.In the preferred version of preparation liposome, use as partially hydrogenated soybean lecithin (Partially hydrogenated soy phosphatidylcholine, PHSPC), polyoxyethylene glycol (the methoxy-terminated polyethyleneglycol of cholesterol (cholesterol), terminal methoxy group, mPEG), (distearoyl phosphatidyl ethanolamine DSPE) is raw material to thanomin di(2-ethylhexyl)phosphate stearate.
In a preferred examples of the present invention, contain the polyoxyethylene glycol-thanomin di(2-ethylhexyl)phosphate stearate of partially hydrogenated soybean lecithin (PHSPC), cholesterol, terminal methoxy group in the described liposome, content ratio is 50～60: 30～40: 5～10.
Can adopt various methods well known to those skilled in the art to carry out the preparation of liposome.The preferred ethanol injection method that adopts is packed modification nucleotide, and prepared liposome has stable in vivo and characteristics such as cycling time is long.
The present invention utilizes and measures cytopathy inhibition method has carried out suppressing viral growth to modification nucleotide activity experiment.Experiment confirm, modification nucleotide provided by the present invention all presents stronger antiviral effect, and this effect is a wide spectrum.
The inventor also utilizes the fluorophotometric method that modification nucleotide has been carried out cell toxicity test, and the result shows that modification nucleotide anti-virus formulation provided by the invention has antiviral specificity, and to the basic nontoxicity of host cell.
Confirm that through clinical experiment modification nucleotide of the present invention also has satisfied curative effect to the patient of virus infectiones such as herpes labialis, reproductive organ bleb.
Can cooperate with various medicine acceptable carriers well known to those skilled in the art, assistant agent with modification nucleotide provided by the invention, be prepared into the antiviral preparation of various formulations.These medicines can be applied to clinical by conventional administering mode.
Described antiviral preparation comprises oral preparation, as tablet, capsule, and injection, externally applied agent, as solution, external-use gel, external-applied ointment also can be made into aerosol etc.Its preparation method can be with reference to the method for conventional medicine formulation preparation.
Embodiment:
The chemosynthesis of embodiment 1 NBC-3
Thymus pyrimidine (5.6 gram) is dissolved in the anhydrous propionitrile, behind the coevaporation, is dissolved in again in 85 milliliters of anhydrous methylene chlorides.With 12.67 gram normal-butyl cyano ethyl-N, (n-butylcyanoethyl-N N-diisopropylphosphoramidite) adds in the thymus pyrimidine solution N-di-isopropyl phosphamide again.Behind violent mixing, add 3.32 grams (Tetrazole), stirred overnight.Reaction mixture after (10 ℃) cooling, slowly adds the decane solution (5～6M) of 10ml t-butylhydroperoxide in the salt ice bath.(about 2～3 hours) changed reaction mixture over to separating funnel after question response was finished, and washed twice with 50 milliliters of methylene dichloride that contain 5% sodium disulfide, washed once with 50 milliliters of saturated sodium-chlorides again.After treating the emulsion layering, tell organic phase.
Organic phase after filtering, with the rotary vacuum drier methylene dichloride that volatilizees.Wash butyraceous decane (3 * 25 milliliters) with ether again, and carefully outwell the upper strata ether.Again oil phase is dissolved in (12.5 milliliters) behind the methylene dichloride, adds 125 milliliters of ether.Through after about 15 minutes quiet putting, outwell solvent, and dry in a vacuum oil phase.
Oil phase with drying is dissolved in methyl alcohol (every gram oil adds 10 ml methanol) at last, and adds 2 normal NaOH, and at room temperature stir about is 1～2 hour, and reaction mixture is removed residual moisture with dried methyl alcohol coevaporation behind vacuum concentration, make product become colloidal substance.Again with propionitrile with this colloid solidification (100 milliliters, about 1～2 hour), use the vacuum-drying solid then.This solid is chest pyrimidine-3 '-5 ' bis phosphoric acid butyl ester (Thyamidine-3 '-5 '-bis-butyl phosphate).
This product warp
31P NMR and
1H NMR analyzes, and its purity is greater than 99%.
Embodiment 2 NBC-3's is protonated
The protonated of NBC-3 can be finished by using acid solution.These acid solutions comprise phosphoric acid, nitric acid, hydrochloric acid or acetate.In the present embodiment, the HCl of 0.1N joins that (every milliliter of 100～300A260 unit) reaches 1～4.5 up to PH in the NBC-3 solution.
NBC-3 solution after the acidification is removed excessive acid and salt through column chromatography (acid resistant form BioRad or P4 post).Through lyophilize, obtain protonated NBC-3 again.
When this compound dissolution during in water or physiological saline, the PH of solution should be in 1～4 scope.
The preparation of embodiment 3 NBC-3 liposomes
The preparation method adopts the ethanol injection method.At first with PHSPC, cholesterol and mPEG-DSPE were dissolved in (the lipid final concentration is 150mM, and ethanol content is about 30%) in the ethanol by 56: 38: 6, were stirred to clear solution.
On the vortex oscillator, slowly lipoprotein solution is joined (50mg/ml) in the NBC-3 solution.The ratio of lipoprotein solution and NBC-3 solution is 1: 2 (v/v).Mixture forms liposome through the polycarbonate membrane press filtration of 200mm.Liposome solutions is again through supersound process 20 seconds, and freeze thawing four times (freezing 5 minutes, melted 5 minutes) is last, removes ethanol through dialysed overnight.Through the Sepharose post liposome and unpacked free NBC-3 branch are opened again.
Between 60～110nm scope, the medicine fat ratio of NBC-3-5 lipid is 1: 4 with the liposome particles size of this method preparation.
The mensuration of embodiment 4 modification nucleotide antiviral effect
The activity utilization of modification nucleotide measure cytopathy inhibition method carry out (Snoeck R, Sakuma T, Clercy ED, Rosenberg A.Holy, Antimicrob.Agents Chemother 1988,32,1839-1844).In 96 orifice plates, hel cell 2-4 CCID
5O(cell cultures infective dose) infects.Through two hours at 37 ℃ down after the absorption, sucking-off is viral adsorption not, adds the nutrient solution (100 microlitre) of the MEM+2% bovine serum that contains modification nucleotide.Experiment is established two holes to every kind of dosage and is repeated.After 6 days, utilize the method for observation of cell pathology (CPE) through 37 ℃ of cultivations, the record antiviral effect.Cell is at first fixed with 70% ethanol, and then with 2.5%Giemse dyeing 2 hours, it is inferior to give a baby a bath on the third day after its birth with distilled water at last, and at room temperature dry.So far, nutrient solution is examined under a microscope cytopathy.Antiviral effect EC
5OExpression (suppressing the required drug level of 50% cytopathy).The results are shown in following table:
| Medication name | Antiviral effect (EC 5O，μg/ml) | |||
| ????HIV-1 | ????HSV-1 | ????HSV-2 | ????HCMV | |
| ????NBC-2 | ????30.1 | ????39.2 | ????41.3 | ????20.6 |
| ????NBC-3 | ????61.0 | ????31.2 | ????32.1 | ????18.4 |
| ????NBC-4 | ????49.2 | ????50.6 | ????39.2 | ????25.7 |
From last table result as seen, modification nucleotide provided by the present invention all presents stronger antiviral effect, and this effect is a wide spectrum.
The cell toxicity test of embodiment 5 modification nucleotides
In order to measure modification nucleotide whether host cell is had cytotoxicity, experiment utilizes the fluorophotometric method, and the amount that hel cell is sucked propidium iodide (PI) is measured.If cell is in state of health, then there is not the suction of PI, dead if cell is tending towards, a large amount of PI then occur and enter cell.3000 hel cells are inoculated in the hole of 96 orifice plates (100 microlitre NEM+10% bovine serum).After 24 hours, in every hole, add medicine through serial dilution.Continue to cultivate after 3 days, every hole adds 100 microlitre PI solution (every milliliter of nutrient solution 40 micrograms).Under the black out condition, be incubated after 60 minutes, the PI flush away will dissociate.With fluorophotometer participating in of PI measured at last.Cytotoxicity CS
5ORepresent (suppressing the required drug level of 50% cell growth).The results are shown in following table:
| Medicine | Cytotoxicity (EC 5O，μg/ml) |
| ????NBC-2 | ????＞100 |
| ????NBC-3 | ????＞100 |
| ????NBC-4 | ????＞100 |
Above result shows that modification nucleotide anti-virus formulation provided by the invention has antiviral specificity, and to the basic nontoxicity of host cell.
The clinical experiment result of embodiment 6 NBC-3 anti-virus infections
Case is selected: carried out the clinical experiment that anti-herpesvirus infects with NBC-3,5 routine experimenters all have bleb in various degree, and its gradient of infection is determined by the specialist.
The experiment medicine: the NBC-3 spray agent of liposome packing, specification is 20 A260 units/ml
Experimental technique: spray medicine 3 time in the affected part every day, each 100 microlitres; Check and the record result of treatment by the specialist after 2 weeks; Gradient of infection is divided into 10 grades by weight, and wherein 1 is the lightest level, and 10 are heavy duty.
Experimental result: after the experimenter local application, all obviously alleviated gradient of infection, concrete outcome sees the following form:
| Case number | Patient | Sex | Age | Disease | Gradient of infection before the treatment | Treatment postoperative infection degree |
| ????40 | ??C.R. | The man | ??36 | Herpes labialis | ????5 | ????1 |
| ????41 | ??H.S. | The woman | ??40 | Herpes labialis | ????4 | ????1 |
| ????42 | ??E.B. | The woman | ??11 | Herpes labialis | ????3 | ????1 |
| ????43 | ??M.K. | The man | ??30 | Herpes labialis | ????6-7 | ????1 |
| ????44 | ??C.W. | The woman | ??57 | The reproductive organ bleb | ????10 | ????1-2 |
Claims (10)
1. antiviral compound, its structure is
Wherein, X and Y are selected from straight chained alkyl, hydroxyalkyl, alkoxyalkyl;
Z is selected from H, replacement or unsubstituted pyrimidyl, replacement or unsubstituted purine radicals;
A is selected from H, alkyl, alkoxyl group, alkoxyalkyl, aryl, thiazolinyl, alkyl alcohol, phenylol, enol.
2. the described antiviral compound of claim 1 is protonated compound; Wherein X and/or Y are selected from normal-butyl or 4-hydroxyl normal-butyl; Z is selected from thymine base or H; A is selected from H or methoxyl group.
3. the described antiviral compound of claim 1, wherein X and Y are CH
3CH
2CH
2CH
2-, Z is a thymine base, A is H.
4. the described antiviral compound of claim 1, feature is to exist with the liposome form.
5. the described antiviral compound of claim 4 contains one or more of following substances in the described liposome; Neutral lipid, cholesterol, polyoxyethylene glycol; Liposome Chinese medicine fat ratio is 1: 9～1: 3; The liposome particles size is 60～110nm.
6. the described antiviral compound of claim 5, described liposome particles size is 80nm～100nm; Medicine fat ratio is 1: 5～1: 3.
7. the described antiviral compound of claim 5, contain the polyoxyethylene glycol-thanomin di(2-ethylhexyl)phosphate stearate of partially hydrogenated soybean lecithin (PHSPC), cholesterol, terminal methoxy group in the described liposome, content ratio is 50～60: 30～40: 5～10.
8. the described antiviral compound of claim 7, the polyoxyethylene glycol of partially hydrogenated soybean lecithin (PHSPC), cholesterol, terminal methoxy group in the described liposome-thanomin di(2-ethylhexyl)phosphate stearate content ratio is 56: 38: 6, granular size is 80nm～100nm, and medicine fat ratio is 1: 4.
9. an anti-viral pharmaceutical compositions contains the described antiviral compound of claim 1～8.
10. use the purposes of the described compound antiviral of claim 1～8.
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|---|---|---|---|
| CN 200510064329 CN1687101A (en) | 2005-04-14 | 2005-04-14 | Nucleotide-like antiviral medicine |
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|---|---|---|---|
| CN 200510064329 CN1687101A (en) | 2005-04-14 | 2005-04-14 | Nucleotide-like antiviral medicine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11851654B2 (en) | 2018-03-19 | 2023-12-26 | National University Corporation Tokyo Medical And Dental University | Nucleic acid with reduced toxicity |
-
2005
- 2005-04-14 CN CN 200510064329 patent/CN1687101A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11851654B2 (en) | 2018-03-19 | 2023-12-26 | National University Corporation Tokyo Medical And Dental University | Nucleic acid with reduced toxicity |
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