CN1118140A - Oligonucleotides modified to improve stability at ACID pH - Google Patents

Oligonucleotides modified to improve stability at ACID pH Download PDF

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Publication number
CN1118140A
CN1118140A CN94191223A CN94191223A CN1118140A CN 1118140 A CN1118140 A CN 1118140A CN 94191223 A CN94191223 A CN 94191223A CN 94191223 A CN94191223 A CN 94191223A CN 1118140 A CN1118140 A CN 1118140A
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oligomer
nucleoside
key
methyl phosphonate
alkyl
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P·S·米勒
P·O-P·佐
L·N·西蒙
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Johns Hopkins University
Genta Inc
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Genta Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Abstract

Acid resistant Oligomers suitable for oral administration, orally acceptable formulations of such Oligomers and preparation of pharmaceutical formations of such Oligomers are provided.

Description

For improving oligonucleotide in the stability modification of acid pH
Background of invention and preamble
The present invention relates to provide the method that demonstrates the oligomer that has improved the stability when acid pH, relate to the method for such oligomer being delivered to its site of action, and relate to it and be used for peroral administration prescription or other helps acid proof dosage form.
The existing report of the depurination of DNA (deoxyribonucleic acid) under acid condition (DNA) (the nucleoside unit loses purine bases by the fracture of glycosidic inkage between base and the glycosyl).(Hevesi，L.，et?al.，J.Amer.Chem.Soc.，94，4715-4720(197))。Treatment Prociclide oral delivery, under normal drug conveying condition, may need to make this drug exposure to reach about 4 hours, and under the slow releasing pharmaceutical transport condition, (for example, consult U.S. Patent No. 4 in stomach acid condition (about pH1), 839,177) reach about 12 hours.Because its deficient in stability under acid condition, thereby can not there be the oligomeric Deoxydization nucleotide of enough oral administrations to keep original drug effect.Ribonucleic acid (RNA) has been in the news more stable than its DNA homologue significantly for the depurination under acid condition, it is reported, this be because on the glycosidic inkage between glycosyl and the base 2 '-obvious stabilizing effect (Hevesi, the L. of hydroxyl, et al. sees above).
Although RNA can resist the depurination under the acid condition, it has limited its treatment serviceability to the sensitivity that is present in the biomaterial, ubiquitous nuclease.In addition, oligoribonucleotide also is infeasible with its unmodified form as drug use, this be since this molecule to the cause of neutrality to the inherent instability of weak basic condition.
Regrettably, it is impossible replacing the phosphoric acid ester bond to protect RNA not to be subjected to the nuclease effect with methyl phosphonate because glycosyl 2 '-hydroxyl ruptures the methyl phosphonate main chain rapidly.
Summary of the invention
The present invention relates to provide the method for the unitary oligomer of nucleoside that comprises preliminary election Base sequence with acidproof form.According to one aspect of the present invention, these oligomer are provided, wherein there is a glycosyl nucleoside unit, promptly 2 '-O-alkyl ribosyl group.Be preferably, this oligomer is neutral on substantially.Better be the oligomer that key between the methyl phosphonate nucleoside is arranged, be more preferably that key is the methylphosphonic acid ester bond between about 50%～about 100% nucleoside.Preferably 2 '-O-alkyl ribosyl group comprises 2 '-methylribose base group.
According to another aspect of the present invention, the oligomer that provides with acidproof form comprises key between the methyl phosphonate nucleoside, the 50%～about 100% methylphosphonic acid ester bond of better having an appointment.
According to yet another aspect, the present invention relates to prepare a kind of method that comprises the unitary oligomer of nucleoside with preliminary election Base sequence, this oligomer is suitable for oral administration and demonstrates acidproof degraded.According to an aspect, this method comprise with have a glycosyl promptly 2 '-O-alkyl ribosyl group, be more preferably 2 '-synthetic this oligomer in nucleoside unit of O-alkyl ribosyl group.Be preferably, this oligomer is neutral on substantially.Be more preferably, synthetic have a unitary oligomer between the methyl phosphonate nucleoside.
In addition, this method comprises that use has synthetic this oligomer in nucleoside unit of key between the methyl phosphonate nucleoside.Good especially is the unitary oligomer between 50%～about 100% methyl phosphonate nucleoside of having an appointment.
On the other hand, the present invention relates to a kind of oligomer of a kind of oral delivery and be used for the treatment of the method for purpose for a kind of mammal, wherein said oligomer comprises a kind of nucleoside unit that purine bases are arranged, and this method comprises a kind of administration of acidproof oligomer.According to this specific embodiments on the one hand, this acidproof oligomer comprises the nucleoside unit, that its glycosyl comprises is a kind of 2 '-O-alkyl ribosyl group, better be a kind of 2 '-O-methylribose base group.Be preferably, this oligomer is neutral on substantially.Acidproof preferably oligomer includes the oligomer of key between the methyl phosphonate nucleoside, is more preferably, and key is the methylphosphonic acid ester bond between about nucleoside of 50%～about 100%.According to a good especially aspect, this acidproof oligomer is with the administration of a kind of controlled velocity releasing pattern.
Except that other factors, the present invention is based on our surprising discovery: by synthetic make it to include a glycosyl promptly 2 '-the unitary oligomer of nucleoside of O-alkyl ribosyl group, the oligomer that perhaps comprises key between the methyl phosphonate nucleoside demonstrates favourable acidproof catalysis depurination and hydrolysis subsequently.
Also believe, include one 2 '-the nucleoside unit of O-alkyl ribosyl group and methyl phosphonate nucleoside between key oligomer obviously with a kind of RNA target molecule generate more stable doublet (duplexes) rather than generation corresponding 2 '-deoxidation-ribonucleotide methyl phosphonate.
According to a further aspect, the present invention relates to comprise a kind of pharmaceutical composition that is the acidproof oligomer of the present invention of controlled velocity releasing pattern.In addition, provide the pharmaceutical composition that comprises a kind of acidproof oligomer, these compositionss itself are tart or are making or lay up period can be exposed to acid condition.
Definition
As used herein, following term has following meanings, is not such unless clearly state.
" purine " or " purine bases " this term not only comprises naturally occurring adenine and guanine, and comprise the modifier of these alkali, the alkali that replacement is for example arranged on 8, or the guanine analog of modification on 6, or adenine analog, 2-aminopurine, and the purine analogue of the nitrogen of carbon on having replaced 9, for example 9-deazapurine derivatives and other purine analogue.
" nucleoside " this term comprises the nucleoside unit and can use with its exchange, means a kind of nucleic acid subunit that comprises a kind of 5 carbon sugar and a kind of nitrogenous base.This term comprises that not only those are with A, G, C, T and the U nucleoside unit as its base, and comprise natural analog and the modified form that has base, comprise pyrimidine analogue, for example false iso-cytosine and pseudouracil and other modification base (for example purine of 8-replacement).In RNA, 5 carbon sugar are ribose; In DNA, it is a kind of 2 '-deoxyribose.This term of nucleoside also comprises other analog of this class subunit, comprise those have the modification glycosyl for example 2 '-analog of O-alkyl ribose.
" phosphonate ester " this term means group
Figure A9419122300081
, wherein R is hydrogen or an alkyl or aryl.The alkyl or aryl group that is suitable for comprises that those spatially do not hinder this phosphine ester bond or group interact with each other.Phosphonate groups both can also can exist by " S " configuration by " R " configuration.Phosphonate groups can be used as and connects phosphorio group key (or link) between the unitary nucleoside of each nucleoside.
" di-phosphate ester " or " diester " means group
Figure A9419122300082
, wherein phosphodiester group can be used as and connect phosphorio group key (or link) between the unitary nucleoside of each nucleoside.
" non-nucleoside monomers unit " means that a kind of wherein base, glycosyl and/or phosphorus main chain are by the alternate monomeric unit of other chemical part.
" nucleoside/non-nucleoside polymer " means a kind of polymer of being made up of nucleoside and non-nucleoside monomers unit.
" oligomeric nucleoside " or " oligomer " mean a kind of nucleotide chain that is connected by key between nucleoside, and its length is generally about 4 to about 100 nucleoside, but its length can be greater than about 100 nucleoside.They are normally synthetic from nucleoside monomers, but also can obtain with enzyme process.Therefore, " oligomer " this term means a kind of oligomeric nucleotide chain that connects each nucleoside monomers with key between nucleoside, thereby comprise oligonucleotide, the oligomeric nucleoside alkyl of nonionic-and aryl-phosphonate analogs, alkyl-and aryl-Thiophosphonate, the thiophosphate of oligonucleotide or dithio acid esters are like thing, the phosphoramidic acid ester analogs of oligonucleotide, the oligomeric nucleoside analog of neutral phosphate, for example phosphotriester and other oligomeric nucleoside analog and the oligomeric nucleoside of modification also comprise nucleoside/non-nucleoside polymer.This term also comprises the nucleoside/nucleotide polymer, and wherein for example one first and second acetal (formacetal) key, sulfo-first and second acetals (thioformacetal) key, a sulfamic acid ester bond or an amino-formate bond replace the group of the one or more phosphorio between each monomeric unit key by a non-phosphorus key.It also comprises nucleoside/non-nucleoside polymer, and wherein all replaced the or modification of glycosyl and phosphorus part becomes morpholino base analogue or polyamide base analogue.It also comprises nucleoside/non-nucleoside polymer, the wherein base of non-nucleoside, glycosyl and phosphate ester main chain or substituted by a non-nucleoside moiety, and perhaps one of them non-nucleoside moiety is inserted in this nucleoside/non-nucleoside polymer.Randomly, described non-nucleoside moiety can be used for connecting the micromolecule that other can interact with target sequence or can change the target cell intake.
" alkyl-or aryl-phosphonate oligomers " this term mean at least one alkyl-or aryl-phosphonate ester nucleoside between the oligomer of key.The alkyl that is suitable for-or aryl-phosphonate groups comprise and spatially do not hinder phosphonate bond or alkyl or aryl group interact with each other.Alkyl group comprises 1 low-grade alkyl group to about 6 carbon atoms of having an appointment preferably.The aromatic yl group that is suitable for has at least one that the ring of conjugate elements electronic system is arranged and comprises isocyclic aryl and the heterocyclic aryl group, and these groups can randomly have substituent group and better have up to about 10 carbon atoms.
This term of " methylphosphonic acid ester oligomer " (or " MP-oligomer ") means the oligomer of key between at least one methyl phosphonate nucleoside.
" middle oligomers " this term means key between the nonionic nucleoside the is arranged oligomer of (key that does not promptly have cation electric charge or anion electric charge) between each nucleoside monomers, and comprise that (for example) has between nucleoside key such as alkyl-or aryl-phosphonate bond, alkyl-or aryl-thiophosphate, neutral phosphate key such as tricresyl phosphate ester bond, particularly neutral triethyl key, and the oligomer of key such as sulfamic acid ester bond, morpholino key, first and second acetal bonds, sulfo-first and second acetal bonds and amino-formate bond between non-phosphorous nucleoside.Randomly, middle oligomers can comprise a kind of conjugate between a kind of oligomeric nucleoside or nucleoside/non-nucleoside polymer and a kind of second molecule that comprises the conjugation partner.Such conjugation partner can comprise that bonding material, lipophilic agent, the nucleic acid modification group of intercalator, alkylating agent, cell surface receptor comprise photocrosslinking agent such as psoralen and can make the group etc. of the target part fracture of nucleic acid.Such conjugation partner can further improve the picked-up of this oligomer, improves the interaction of this oligomer and target sequence, or changes the pharmacokinetics distribution of this oligomer.Basic demand is that oligomeric nucleoside that this oligomer conjugates comprised or nucleoside/non-nucleoside polymer is neutral substantially.
" neutral substantially " this term means that those keys between the nucleoside between its each nucleoside monomers are the oligomer of nonionic key at least about 80% with regard to oligomer.
" neutral alkyl-or aryl-phosphonate oligomers " this term means that key between its neutral nucleoside comprises at least one alkyl-or the middle oligomers of arylphosphonic acid ester bond.
" neutral methylphosphonic acid ester oligomer " this term means that key between its nucleoside comprises the middle oligomers of at least one methylphosphonic acid ester bond.
" acidproof " this term means the oligomer that more can resist acid catalysis N-glycosyl bond hydrolysis depurination than the oligomeric nucleoside of deoxyribose.
" triplet " (triplet) or " triad " (triad) this term mean the hydrogen bond coordination compound of the base of three kinds of nucleoside between a kind of base (if bifilar target) of a kind of base of a kind of a kind of base (if sub-thread chain) of target sequence or multiple base (if double-stranded chain), second strand of chain and the 3rd strand of chain (if sub-thread target sequence) or the 3rd strand of chain.
Fig. 1 explanation comprises RNA, DNA, a kind ofly has 2 '-the complete adenine percentage rate of the oligomer of the unitary methylphosphonic acid ester oligomer of O-methylribose base and a kind of methylphosphonic acid ester oligomer change curve in time.
Fig. 2 explanation is as the complete adenine percentage rate logarithm of the identical oligomer of mapping among Fig. 1 change curve in time.
Fig. 3 explanation comprises RNA, DNA, a kind ofly has 2 '-oligomer of the unitary methylphosphonic acid ester oligomer of O-methylribose base and a kind of methylphosphonic acid ester oligomer is at 37 ℃ depurination percentage rate change curve in time.
Fig. 4 explanation as the identical oligomer of mapping among Fig. 3 at 37 ℃ depurination percentage rate logarithm change curve in time.
Fig. 5 illustrates a kind ofly has 2 '-the unitary methylphosphonic acid ester oligomer of O-methylribose is at the complete main chain percentage rate of 37 ℃ and pH1 change curve in time.
Fig. 6 explanation comprises a kind of oligomeric DNA (deoxyribonucleic acid) (1), a kind ofly has 2 '-the unitary methylphosphonic acid ester oligomer of O-methylribose (2) the and a kind of oligomer of the unitary methylphosphonic acid ester oligomer of deoxyribose (3) and a kind of melting curve of DNA target hydridization arranged.
Fig. 7 explanation is with the same oligomer of Fig. 6 and a kind of melting curve of RNA target hydridization.
Fig. 8 illustrates the dimer example, comprise (a) a kind of oligomeric 2 '-O-methylribonucleotide methyl phosphonate, (b) a kind of oligomeric dezyribonucleoside methyl phosphonate and (c) a kind of oligomeric dezyribonucleoside.
The detailed description of invention
Oligomer preferably
Oligomer of the present invention include a glycosyl be one independent select 2 '-the nucleosides unit of O-alkyl ribosyl group. Applicable is the alkyl group of 1 to 5 carbon atom. Particularly preferred nucleosides has one 2 '-O-methylribose base group.
Oligomer chain with key between selected nucleosides can be familiar with the known synthetic technology preparation of personnel of this gate technique easily according to those. For example, commercial machine, reagent and experimental program (protocols) can be used for the synthetic oligomer that key between di-phosphate ester and some other phosphorous nucleosides is arranged. Also consult Gait, M.J., Oligonucleotide Synthesis:A Practical Approach (IRL Press, 1984); Cohen, Jack S., Oligodeoxynucleotides Antisense Inhibitors of Gene Expression, (CRC Press, Boca Raton, FL, 1989); With Oligonucleotides and Analogues:A Practical Approach, (F.Eckstein, ed., 1991). Have the preparation of the oligomer of key between some non-phosphorous nucleosides to see U.S. Patent No. 5,142,047 for details, its open document is classified this paper list of references as.
It better is neutral substantially oligomer.
According to a particularly preferred aspect, these oligomer have key between the methyl phosphonate nucleosides. Be more preferably, key all is the methylphosphonic acid ester bond between all these nucleosides. There is the oligomer of the mixture of key and other nucleosides key between the methyl phosphonate nucleosides can be used for preferably some treatment indication, thereby should includes in the scope of the present invention.
Be preferably, this oligomer comprises about 4 to about 40 nucleosides, is more preferably about 6 to 30 nucleosides. Particularly preferred is 8 oligomer to about 20 nucleosides of having an appointment.
Effectiveness and administration
Oligomer provided by the invention can with a kind of target sequence a kind of nucleic acid or a kind ofly have the albumen of high selectivity to form a kind of high affinity complex for example. For example, the oligomer of derivatization can be used to a kind of nucleic acid in a target site bonding, then by crosslinked (psoralen) or make one chain or two strands of chains all rupture, make it irreversibly modification. By target site that will rupture of careful selection, can use wherein one chain to cut off one section selected nucleic acid sequences as a molecular scissors single-mindedly.
The oligomer that this paper provides can be derivatized, to introduce a nucleic acid reaction or modified group, can make the target sequence reaction of this group and nucleic acid fragment or one, with irreversibly modification, degraded or destroy this nucleic acid, thereby irreversibly suppress its function.
These oligomer can be used to passivation or inhibition or change a kind of specific gene in the living cells or the expression of its target sequence, so that can selective passivation or inhibition or change and express. This target sequence can be DNA or RNA, for example a kind of before-mRNA or a kind of mRNA. The mRNA target sequence comprises start code subarea, poly-adenosine district, mRNA attach the names of pre-determined candidates position or adhesive bond section. If these oligomer also can be used for permanently passivation, close or destroy those and can produce the gene that defectiveness or undesirable product or activation will cause undesirable effect.
Because oligomer provided here can form dyad or triple helical complex or other and the stable form of associating in transcribed nucleic acid district, these complexs can be used for " antisense " (antisense) or three strands of chains are treated (triple strand therapy). Here used " antisense " treatment is a classification term, is included in isolated test or the live test to come the undesirable DNA of passivation or RNA order with specific bonding oligomer.
The feature of a lot of diseases and other illness is to have undesirable DNA or RNA, and they may be the sub-thread chains in some cases, and may be double-stranded chain in other cases. These diseases and illness can be treated with the technical antisense therapy principle of generally all understanding. Antisense therapy comprises by replenishing or by any other specific adhesive means, by forming dyad or triple helical complex, aiming at specific DNA or a RNA target sequence in situation of the present invention.
The oligomer that uses among the present invention can be individually dosed, perhaps can use the various combinations of oligomer, with for contiguous target or remote target, or for generation of the comprehensive effect of antisense mechanism and aforementioned general mechanism.
On therapeutical uses, these oligomer can be mixed with various administering modes, comprise oral administration, surperficial administration or topical. What possibility was useful is, allow the medicine prescription contain acidproof oligomer, thereby during making, it maybe when making such prescription that to a certain degree acid be arranged itself, can contact acid condition, in the hope of more can compatible agents area such as the acid outer condition in vogue of skin. Technology and prescription generally can be consulted Remington ' s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition. The oligomer active component general with a kind of carrier for example a kind of diluent or excipient composition, this can comprise filler, extender, binding agent, wetting agent, disintegrant, surfactant, erodable polymer or lubricant, because of the character of administering mode and dosage form different. Typical dosage form comprises tablet, and pulvis, liquid preparation comprise suspending agent, emulsion agent and solution, granule, and capsule.
Oligomer of the present invention is particularly suitable for oral administration, may need to make drug exposure to reach about 4 hours in the acid condition of stomach under the common drug transport condition, and reach about 12 hours when carrying with the slowly-releasing form. For the treatment of some illness, it may be favourable preparing these oligomer with the slowly-releasing form. Its open document is classified U.S. Patent No. 4,839,177 this paper list of references, the people such as Colombo as and has been described some preferably controlled velocity delivery system. For oral administration, these oligomer are mixed with oral administration form common and that time-delay discharges, for example capsule, tablet and liquid agent.
These oligomer are especially applicable to the surperficial drug-delivery preparation of preparation, and are acid outer because skin has, and the prescription that contains these acidproof oligomer can prove favourable. According to described in the u.s. patent application serial number 07/707,879 of classifying this paper list of references as, find that these oligomer can see through skin and mucous membrane, this also can be favourable. Also can look to provide the prescription that contains acid medium.
For surperficial administration, the oligomer that uses among the present invention is mixed with technical general known ointment, ointment, eye drops, gel or cream.
The general administration also can be by means of carrying out through the mucous membrane means or through the skin means, and perhaps these compounds can oral administration. For through mucous membrane or percutaneous dosing, in prescription, use the penetrating agent that is suitable for the barrier that will see through. Such penetrating agent generally is technical known, and comprises such as the bile salt and the fusidic acid derivative that are used for mucosal. In addition, can use washing agent to be beneficial to infiltration. Mucosal can be undertaken by using such as nasal spray and the prescription or the suppository that are suitable for inhalation-type drug administration.
For helping to understand the present invention, adopt following example to describe a series of result of experiment.The following example relevant with the present invention should not be understood as that the concrete the present invention that limited certainly, and at present known or development afterwards, belong within this gate technique skilled person scope the present invention like this some variations be considered to fall into as within the claims of the present invention scope after this.
Example
Example 1
2 '-O-methyladenosine synthetic agent synthetic
A.5 '-O-dimethoxytrityl-2 '-O-methyl-3 '-O-
(N, N-diisopropylaminoethyl-O-beta-cyano ethyl phosphine)-N-benzoyl
Synthesizing of adenosine
5 '-O-dimethoxytrityl-2 '-O-methyl-N-benzoyl adenosine (0.75g; 1.09 mM) (Barry Associates is Inc.) with anhydrous 1/1 acetonitrile/diisopropylethylamine-play coevaporation 3 times.Then this nucleoside is dissolved in the 30ml anhydrous acetonitrile.Add diisopropylethylamine (0.570ml in room temperature; 3.27 mM: 3 equivalents) (Aldrich), add chloro-N subsequently, N-diisopropylaminoethyl-beta-cyano ethyoxyl phosphine (0.386ml; 1.64 mM; 1.5 equivalent) (ABN).-hour after, reaction is finished, as use 50/45/5 ethyl acetate/hexane/triethylamine as the silica gel plate thin layer chromatography (TLC) of eluant determined.Evaporating solvent, residue are dissolved in the dichloromethane, with the saturated aqueous solution of sodium bicarbonate washing, use the anhydrous magnesium sulfate drying organic layer.Crude mixture is purification on handling with the silicagel column of its silica gel acidity that neutralizes with triethylamine in advance.Product is with 50/49/1 ethyl acetate/hexane/triethylamine eluting.With pure fraction collection, drying, obtain 440mg (0.45 mM: product 41.4%).
B.5 '-O-dimethoxytrityl-2 '-O-methyl-3 '-O-
The preparation of (N, N-diisopropylaminoethyl-methylphosphine)-N-benzoyl adenosine
5 '-O-dimethoxytrityl-2 '-O-methyl-N-benzoyl adenosine (1.0g; 1.45 mM) (Barry Associates is Inc.) with anhydrous 1/1 acetonitrile/diisopropylethylamine coevaporation 3 times.This nucleoside is dissolved in the 20ml anhydrous acetonitrile.Add diisopropylethylamine (1.11ml in room temperature; 6.4 mM: 4.4 equivalents) (Aldrich), add chloro-N subsequently, N-diisopropylaminoethyl-methylphosphine (0.582ml; 3.2 mM; Equivalent) 2.2 (JBL Scientific).Afterreaction was complete in one hour, as use 50/45/5 ethyl acetate/hexane/triethylamine as the silica gel plate thin layer chromatography of eluant determined.Evaporating solvent, residue are dissolved in the dichloromethane, with the saturated aqueous solution of sodium bicarbonate washing, use the anhydrous magnesium sulfate drying organic layer.Crude mixture is purification on handling with the silicagel column of its silica gel acidity that neutralizes with triethylamine in advance.Product is with 50/49/1 ethyl acetate/hexane/triethylamine eluting.With pure fraction collection, drying, obtain 340mg (0.41 mM; 28%) product.
Example 2
A kind of preparation that the deoxyadenosine tetramer (chemical compound 1) of key between the di-phosphate ester nucleoside is arranged
This tetramer is to use a kind of standard phosphoramidate step of Milligen 8750 DNA synthesizers (to consult such as Gait; M.J.; Oligonucleotide Synthesis:APractical Approach, 1984 (IRL Press)) synthetic and deprotection.This chemical compound of reversed-phase high-performance liquid chromatography (HPLC) purification with acetonitrile (" the ACN ") gradient in Whatman RACII analytical column and the 0.1M second triethylenetetraminehexaacetic acid ammonium (0-30%ACN, 40 minutes, flow was 1ml/ minute).
Example 3
A kind of deoxyadenosine methyl phosphonate tetramer (chemical compound 2) and a kind of 2 '-preparation of the O-methyladenosine methyl phosphonate tetramer (chemical compound 4)
Chemical compound 2 be with 5 '-(dimethoxytrityl) adenosine-3 '-[(N, N-diisopropylaminoethyl) methyl] phosphine ammonia ester (phosphonoamite) monomer is synthetic.Solid phase synthesis is to carry out on the methacrylate polymers supporter according to the suggestion of manufacturing firm with a kind of Biosearch Model 8750 DNA synthesizers, and different is to have done following improvement: monomer is dissolved in the acetonitrile with the concentration of 100mM: 2.5% dichloroacetic acid in deblocking reagent=dichloromethane; Oxidant reagent=every liter contains 25g iodine: 10% acetic anhydride in the CAP A=acetonitrile by 2.5% water, 25%2 in the solution that 6-lutidines and 72.5% oxolane are formed; 0.625%N in the CAP B=pyridine, the N-dimethyl aminopyridine; Coupling time lengthening to 4 minute.
When end of synthesis, remove the dimethoxytrityl group on the oligonucleotide.
Then oligonucleotide cracking from the supporter is got off, and make it deprotection.From synthetic tube, taking out, put in 1 drachm (dosage unit made in Great Britain) glass vial that screw top arranged with the bonded oligonucleotide of supporter.Supporter is at room temperature 1ml acetonitrile/ethanol/NH 4OH (9/9/1) solution-treated 30 minutes.Then, in reaction vessel, add the 1ml ethylenediamine, allow reaction carry out 6 hours until finishing.Then, take out the supernatant that contains oligomeric nucleoside in this supporter, this supporter is with 2ml 1/1 acetonitrile/water rinsing 2 times, this moment, rinsing liquid and supernatant merged.It is 30ml that the solution with water that merges is diluted to cumulative volume, the about 4ml 6N of reuse HCl neutralization.Neutral solution is used 10ml acetonitrile, 10ml 50% acetonitrile/100mM bicarbonate three second ammoniums and 10ml 25mM bicarbonate three second ammonium pre-equilibrations before this successively with Waters C-18 Sep-Pak post desalination, this post.Allowing after reaction solution passes through this post, with the 30ml water washing it.Use 5ml 1/1 acetonitrile/water eluting product then.
Oligonucleotide is the high-efficient liquid phase chromatogram purification on the reversed-phase column (Whatman RACII) of acetonitrile gradient in using 50mM second triethylenetetraminehexaacetic acid ammonium.
Chemical compound 4 as to 2 of use-case 1 (B) '-the monomeric chemical compound 2 of O-methyladenosine as described in synthetic, deprotection and purification, different is, coupling time lengthening to 3 minute so that spatial obstacle bigger 2 '-the fully coupling of O-methyl monomer reagent.Chemical compound 4 be combine on the supporter of deoxyadenosine synthetic.
Example 4
The preparation of a kind of nucleoside oligoribonucleotide tetramer (chemical compound 3)
The oligomeric ribonucleotide tetramer (chemical compound 3) be with 5 '-O-dimethoxytrityl-2 '-O-t-butyldimethylsilyl-3 '-O-N, N-diisopropyl-beta-cyano ethylamino phosphite ester adenosine (Millipore) is synthetic.Synthesize and carry out with micromole's scale, adopt a kind of Milligen 8750 automated DNA synthesizers, the amino phosphite ester step of use standard Milligen, different is, coupling time lengthening to 12 minute, make spatial obstacle bigger 2 '-O-t-butyldimethylsilyl RNA monomer has time response more fully.Synthetic be combining 2 '-begin on the control cellular glass (Pennisula laboratory) of O-t-butyldimethylsilyl adenosine.All other oligomeric nucleoside synthetic agents are all described in Milligen standard test scheme.After synthetic, oligomeric ribonucleotide sterilization, dispose under the no RN enzyme condition.Water is handled the sterilization of steaming and decocting under high pressure subsequently of spending the night with 0.5% pyrocarbonic acid diethyl ester.All glass drying ovens are all 300 ℃ of bakings at least 4 hours.Oligomeric nucleoside is that earlier this combines supporter 15 hours of oligomer and deprotection and from the supporter under the cracking 55 ℃ of processing with 3/1 ammonium hydroxide/ethanol.The supernatant decantation that will contain oligonucleotide then comes out and it is evaporated to dried.Then, resulting residue (Aldrich) was handled 24 hours with 0.6ml 1M tetrabutylammonium fluoride tetrahydrofuran solution (moisture 5% or following) in room temperature.Add 0.6ml 2M second triethylenetetraminehexaacetic acid aqueous ammonium (pH7) and make reaction terminating.The desalination of reactant mixture allows this solution carry out with sterilized water by a post 10DG (BiO-Rad).Make the oligonucleotide drying of desalination then.Chemical compound (is seen example 2) as described in to chemical compound 1 purification on high performance liquid chromatography.
Example 5
A kind of have 2 of key between the di-phosphate ester nucleoside '-the tetrameric preparation of O-methyladenosine oligonucleotide
This oligonucleotide is synthetic as described in to chemical compound 1, deprotection and purification, and different is, coupling time lengthening to 4 minute so that spatial obstacle bigger 2 '-the fully coupling of O-methylating reagent.This chemical compound be combine on the supporter of deoxyadenosine synthetic.
Example A
The absolute acid stability of oligomer is measured
Measured the relative stability of chemical compound 1 to 5 under the acid condition of simulation stomach pH.These chemical compounds are handled with the HCl aqueous solution of pH1, determine sugared bond fission speed over time by the appearance of clearing up the product adenine on the reversed-phase HPLC chromatogram.
These reversed-phase HPLC analyses are to carry out with a Beckman System Gold HPLC who is equipped with 126 type pumps, 168 type photodiode array detectors and 507 type automatic samplers.(100 * 4.6mm) analytical columns are analyzed with a Whatman RAC II ODS 3.Used solvent system is the acetonitrile gradient in the 0.1M second triethylenetetraminehexaacetic acid aqueous ammonium of pH7.This gradient is to be 0 to 2% acetonitrile in 10 minutes, is 2 to 60% acetonitriles subsequently in other 10 minutes.This gradient is returned 0% acetonitrile then, so that this column equilibration is prepared sample introduction next time.Flow velocity is 1ml/ minute.This gradient is clearly differentiated the various by-products that adenine and initial reagent and acid are cleared up.
With sterilized water (seeing above-mentioned sterilization experimental program) preparation a kind of 100ml 0.1M HCl (pH1.05) stock solution, carry out HPLC and analyze, removed baseline interference to guarantee the adenine elution zone.Baseline is clean in desirable zone.
Then, make the 10.D. unit by injecting 100 μ l 260These tetrameric depurination speed is determined in dissolving, to obtain zero hour time point.After this, perhaps press 0.5 hour interval (chemical compound 1 and 2) or press 1 hour (chemical compound 3 to 5) at interval, gather 10 μ l samples automatically.The results are shown in following table I.
Fig. 1 illustrates the complete adenosine percentage rate of these tetramers time history plot.Fig. 2 is complete adenosine percentage rate logarithm time history plot.Calculated the K of these tetramer depurination speed from logarithmic curve chart AAnd the half-life.Table I comprises these numerals, and with di-phosphate ester tester (chemical compound 1) depurination relative velocity relatively.
Table I
Compound number Rate constant (second -1) at 20 ℃ ??t 1/2(hour) The relative velocity of depurination
1 (di-phosphate ester) ????374.4 ????6.66 ????1.00
2 (methyl phosphonates) ????70.56 ????35.66 ????0.188
????3 ????(RNA) Can not measure (＜1) Can not measure (＞3000) Can not measure (＜0.001)
4 (2 ' OMe methyl phosphonates) * ????2.16 ????1155 ????0.0057
*To 3 '-terminal deoxyadenosine revises.
These results clearly confirm, 2 '-O-methyladenosine methyl phosphonate compares with the di-phosphate ester deoxyadenosine or compares with the similar thing of methyl phosphonate deoxyadenosine, all will stablize manyly (175 times).2 '-this a small amount of depurination that O-methyladenosine methyl phosphonate shows, be likely since as 3 of deoxyadenosine residue '-terminal nucleoside rather than because unexistent 2 on the supporter '-cause of OMe adenosine.For this species diversity of depurination aspect is described, after pH1 handled 4 hours, about 33% di-phosphate ester deoxyadenosine depurination had only 7% methyl phosphonate deoxyadenosine depurination, and 2 '-O-methyladenosine methyl phosphonate depurination only 0.28%.
A kind of very surprising observation is the tetrameric stability of methyl phosphonate deoxyadenosine.The phosphonate ester main chain is beat all to the stabilizing effect of glycosidic inkage.
Example B
Oligomer is in the mensuration of 37 ℃ absolute acid stability
The relative stability of chemical compound 1 to 4 is to use an electric heating piece to make temperature remain on 37 ℃, by what measure like that described in the embodiment A.
The results are shown in the following Table II.
Table II
Compound number Rate constant (second -1) at 37 ℃ T1/2 (hour) The relative velocity of depurination
1 (di-phosphate ester) ????1800 ????1.39 ????1.00
2 (methyl phosphonates) ????203.9 ????12.24 ????0.114
????3 ????(RNA) Can not measure (＜1) Can not measure (＞3000) Can not measure (＜0.001)
4 (2 ' OMe methyl phosphonates) * Can not measure (＜1) Can not measure (＞3000) Can not measure (＜0.0001)
*To 3 '-terminal deoxyadenosine revises.
Fig. 3 illustrates the depurination speed of the adenosine tetramer pH1.0 and 37 ℃, with the depurination percentage rate time is mapped.Fig. 4 illustrates the depurination speed of the adenosine tetramer pH1.0 and 37 ℃, with depurination percentage rate logarithm the time is mapped.Fig. 5 explanation (2 '-O-methyl A) 3(dA) the methyl phosphonate main chain of methylphosphonic acid ester oligomer is to the stability of the acid condition of pH1.0 and 37 ℃.These conditions be the 0.3mM oligomer in 0.1M HCl, pH1.0 is at 37 ℃.Detectable limit is 0.2%; Error≤2%.Speed is to be determined by the free adenine and the chemical seed total amount recruitment relatively of band adenyl, carries out HPLC subsequently and analyzes.Data to 2 '-depurination and 3 of O-methyladenosine methylphosphonic acid ester oligomer '-terminal deoxyadenosine fracture revises.
Example C
A kind of oligomeric-2 '-absolute acid stability of O-methylribonucleotide methyl phosphonate
With 2 of due care '-O-methylribonucleotide methylphosphine carboxylic acid amide esters synthone prepare a kind of oligomeric-2 '-methylribonucleotide methyl phosphonate (A).In the following symbol of pointing out, Nm represent one 2 '-the O-methylribonucleotide, following line is represented the position of methylphosphonic acid ester bond.5 of this oligomer '-nucleoside between key be a kind of phosphodiester bond.The such deprotection of this oligomer:, use 95% ethanol (volume ratio 1: the 1) solution-treated of ethylenediamine subsequently with the buffered hydrazine hydrate pyridine solution of acetic acid sequential processing.This oligomer is with the DEAE cellulose chromatography, subsequently with the preparation HPLC purification that is equipped with the C-18 reversed-phase column.
r-AU mA mG mG mA mU mU mU mG mU mC????????A
d-ATAGGATTGTC????????????????????????????????B
d-ATAGGATTTGTC???????????????????????????????C
Oligomer A be with polynucleic acid kinases and γ-[ 32P]-ATP carry out phosphorylization.Then, the oligomer of phosphorylization is with 0.1N HCl or 1.0N HCl be incubated overnight at 37 ℃ (～14 hours).Then, this oligomer is analyzed with polyacrylamide gel electrophoresis under the degeneration condition.Do not detect this oligomer degraded under these conditions.According to our experience, dezyribonucleoside methyl phosphonate (B) with 0.1N HCl 37 ℃ be incubated overnight or with 1.0N HCl at 37 ℃ of incubation some hrs, can cause the considerable depurination of G and A nucleoside.The spontaneous hydrolysis of methylphosphonic acid ester bond can take place in this depurination thereupon, causes producing the shorter oligomer of chain length.The experience in past shows, Prociclide as the purine residue in (C) to acid catalysis depurination even more responsive.
Therefore, these result of experiment show, oligomeric-2 '-the oligomeric dezyribonucleoside methyl phosphonate of energy force rate or the corresponding purine nucleosides in the oligomeric dezyribonucleoside of the antiacid catalysis depurination of purine nucleosides in the O-methylribonucleotide methyl phosphonate is much bigger.Oligomeric-2 '-the apparent ability of the antiacid catalysis depurination of O-methylribonucleotide methyl phosphonate has important treatment consequence, especially if these oligomer are peroral administration.
Example D
Oligomeric-2 '-O-methylribonucleotide methyl phosphonate is to the hydridization of complementary DNA and RNA target
Measured the hydridization performance of oligomer 2, and compared with the hydridization performance of oligomer 1 and 3.Hydridization to complementary single-stranded DNA target D or RNA target E is to carry out in the buffer agent that contains 10mM HEPES (pH7.0), 2mM EDTA.Melting curve that obtains and melt temperature be illustrated in Fig. 6 and 7 (annotate: in these figure, N mRepresent a kind of 2 '-the O-methylribonucleotide).Prociclide 1 all forms stable hybrid with DNA target and RNA target.Oligomeric-2 '-O-methylribonucleotide methyl phosphonate 2 not with DNA target hydridization, but form highly stable dyad with the RNA target.This behavior is opposite with the behavior of oligomeric dezyribonucleoside methyl phosphonate 3, and the latter and DNA target form stable dyad, but only forms very weak dyad with the RNA target.These results show, oligomeric-2 '-O-methylribonucleotide methyl phosphonate can form more stable hybrid with RNA than oligomeric dezyribonucleoside methyl phosphonate, and therefore, they are more effective antisense reagent when being target with cellularity or viral RNA.
Table III
Tmd-G?A?C?A?A?A?T?C?C?T?A?T
1 28 ℃ of C U of (DNA palladium) C T G T T T A G G A T A-d *G *U *U *U *A *G *G *A *U *3 30 ℃ of buffer agents of 2＜0 ℃ of C T of A-r G T T T A G G A T A-d: 10mM HEPES pH7.0
2mM?EDTA
Table IV
Tmr-G?A?C?A?A?A?U?C?C?U?A?U
1 18 ℃ of C U of (RNA palladium) C T G T T T A G G A T A-d *G *U *U *U *A *G *G *A *U *3＜0 ℃ of buffer agents of 2 20 ℃ of C T of A-r G T T T A G G A T A-d: 10mM HEPES pH7.0
2mM?EDTA

Claims (30)

1. a kind of method that includes the unitary oligomer of nucleoside of the preliminary election Base sequence that is acidproof form is provided, wherein the nucleoside unit of this oligomer have one and belong to 2 '-glycosyl of O-alkyl ribosyl group.
2. according to the oligomer of claim 1, it is neutral substantially that wherein said oligomer synthesizes.
3. according to the oligomer of claim 2, wherein this oligomer synthesizes and has key between the methyl phosphonate nucleoside.
4. according to the oligomer of claim 3, wherein said alkyl is a methyl.
5. according to the oligomer of claim 4, comprise about 50% to key between about 100% methyl phosphonate nucleoside.
6. preparation method that comprises the unitary oligomer of nucleoside of the preliminary election Base sequence that is suitable for oral administration and demonstrates antiacid degradation capability, comprise with there being one belonging to 2 '-the nucleoside unit of the glycosyl of O-alkyl ribosyl group synthesizes described oligomer.
7. according to the oligomer of claim 6, it is synthesized to being neutral substantially.
8. according to the oligomer of claim 7, wherein said oligomer synthesizes has key between the methyl phosphonate nucleoside.
9. according to the oligomer of claim 8, wherein said alkyl is a methyl.
10. according to the oligomer of claim 9, comprise about 50% to unit between about 100% methyl phosphonate nucleoside.
11. a kind of method that includes the unitary oligomer of nucleoside of the preliminary election Base sequence that is acidproof form is provided, and wherein this oligomer synthesizes and has key between the methyl phosphonate nucleoside.
12., comprise about 50% to key between about 100% methyl phosphonate nucleoside according to the oligomer of claim 11.
13. a preparation method that includes the unitary oligomer of nucleoside of the preliminary election Base sequence that is suitable for oral administration and demonstrates antiacid degradation capability comprises with there being the nucleoside unit of key between the methyl phosphonate nucleoside to synthesize described oligomer.
14., comprise about 50% to unit between about 100% methyl phosphonate nucleoside according to the oligomer of claim 13.
15. a kind of oligomer of oral delivery is used for the method for medical purpose to mammal, described oligomer includes the nucleoside unit of purine bases, and this method comprises a kind of administration of acidproof oligomer.
16. according to the method for claim 15, wherein said oligomer includes one and comprises 2 '-the nucleoside unit of the glycosyl of O-alkyl ribosyl group.
17. according to the method for claim 16, wherein said oligomer is neutral substantially.
18. according to the method for claim 17, wherein said alkyl is a methyl.
19. according to the method for claim 18, wherein said oligomer is a kind of methylphosphonic acid ester oligomer.
20. according to the method for claim 15, wherein said oligomer is neutral substantially.
21. according to the method for claim 20, wherein said oligomer is a kind of methylphosphonic acid ester oligomer.
22. according to claim 15,16, one of 19 or 21 method, wherein said oligomer is with the administration of a kind of controlled velocity delivery mode.
23. being the pharmaceutical composition that the controlled velocity releasing pattern comprises a kind of acidproof oligomer, described oligomer includes one and comprises 2 '-the nucleoside unit of the glycosyl of O-alkyl ribosyl group.
24. according to the oligomer of claim 23, it comprises at least 8 nucleoside unit.
25. according to the method for claim 23, wherein said oligomer includes and comprises 2 '-the nucleoside unit of the glycosyl of O-methylribose base group.
26. according to the method for claim 25, wherein said oligomer is neutral substantially.
27. according to the method for claim 26, wherein said oligomer is a kind of methylphosphonic acid ester oligomer.
28. peroral administration pharmaceutical composition that includes unitary acidproof oligomer between the methyl phosphonate nucleoside, is the controlled velocity releasing pattern.
29. according to the compositions of claim 28, wherein said oligomer includes and belongs to 2 '-the nucleoside unit of the glycosyl of O-methylribose base group.
30. pharmaceutical composition that under acid condition, can keep its therapeutic activity, in the compatibility prescription, comprise the acidproof oligomer for the treatment of effective dose.
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