EP2714708A1 - Dérivés d'adénosine ou de désoxyadénosine modifiés en position 8 et leur procédé de synthèse - Google Patents

Dérivés d'adénosine ou de désoxyadénosine modifiés en position 8 et leur procédé de synthèse

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Publication number
EP2714708A1
EP2714708A1 EP12731710.5A EP12731710A EP2714708A1 EP 2714708 A1 EP2714708 A1 EP 2714708A1 EP 12731710 A EP12731710 A EP 12731710A EP 2714708 A1 EP2714708 A1 EP 2714708A1
Authority
EP
European Patent Office
Prior art keywords
derivative
formula
group
deoxyadenosine
adenosine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12731710.5A
Other languages
German (de)
English (en)
Inventor
Massimo Luigi Capobianco
Maria Luisa NAVACCHIA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consiglio Nazionale delle Richerche CNR
Original Assignee
Consiglio Nazionale delle Richerche CNR
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Filing date
Publication date
Application filed by Consiglio Nazionale delle Richerche CNR filed Critical Consiglio Nazionale delle Richerche CNR
Publication of EP2714708A1 publication Critical patent/EP2714708A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
    • C07H21/04—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 deoxyribosyl as saccharide radical

Definitions

  • the present invention relates to adenosine or deoxyadenosine derivatives suitable for use in the synthesis of oligonucleotides, as well as a method for the synthesis of the above- mentioned derivatives.
  • the chemical synthesis also allows binding to the synthetic oligonucleotides a range of functional groups capable of adding new functionalities to the oligonucleotide, such as, for example, functional fluorescent groups, intercalating residues, or groups binding certain proteins or drugs.
  • the functionalization of the synthetic oligonucleotide is usually done through post- synthesis modifications, or directly on the synthesizer, using phosphoramidites that are modified compared to those normally used.
  • the functionalization on the synthesizer has the advantage of a better chemical control and better guarantees for an efficient purification of the final compound.
  • a range of modified phosphoramidites with about twenty functional substituents is commercially available, in order to obtain e.g.
  • strands with a terminal amino group (-NH 2 ) to address further post-synthesis modifications; with a terminal thiol group (-SH) to allow the anchorage of the oligonucleotide on gold surfaces; with a terminal dabcyl group to be used as a molecule quencher in combination with a fluorescent substituent on the other end of the strand to implement fluorescent probes of the "molecular beacon" type; with biotine to bond avidine; with cholesterol to modify the cellular uptake; with fluorescent substituents to implement probes; with psoralen for the intercalation in the double strand formed with the complementary strand and to bond it covalently after irradiation, etc.
  • a terminal amino group -NH 2
  • -SH terminal thiol group
  • the functional group is directly linked to phosphorous (in such a case, it does not contain the nucleoside base); in other cases, instead, it is linked at the position C8 of purines or the position C6 of pyrimidines. Modifications of the nitrogen bases in these positions generally allow maintaining unaltered the recognition of the complementary strands according to the Watson and Crick bonds. When the base is present, the modification can be inserted into a strand of the synthetic oligonucleotide and optionally incorporated several times in the same filament.
  • Navacchia et al. Macromol. Rapid Commun. 2010, 31, 351-355 discloses the derivatization of 8-Br deoxyadenosine with a short chain dithioalkyl linker, to obtain a deoxyadenosine derivative to be polymerized in the presence of FeCl 3 , thereby obtaining polymers capable of self-organizing that are suitable in applications in the electronics industry.
  • the methods that are available in the state of the art for the synthesis of modified adenosines or deoxyadenosines have the drawback that they need quite complex procedures, and that they fail to obtain high yields.
  • the derivatives of the adenosine modified at position 8 described in the state of the art contain thioalkyl chains that are not longer than 3-5 carbon atoms, which not always allow binding a functional substituent with a sufficient adaptation capacity.
  • Object of the present invention is to overcome the drawbacks of the state of the art mentioned above.
  • adenosine or deoxyadenosine derivatives modified at position 8 having the general formula (I) as defined in the annexed independent claim 1 and by the synthesis method of the above-mentioned derivatives as defined in the annexed independent claim 8.
  • the dependant claims define further characteristics of the derivatives and the method for the synthesis thereof, and are an integral part of the description.
  • the synthesis method of the adenosine or deoxyadenosine derivatives modified at position 8 which is the object of the invention has the advantage of achieving high yields and requiring procedures that are less complex than those described in the state of the art to obtain other adenosine derivatives. Furthermore, the derivatives of adenosine modified at position 8 obtained by the method of the invention are advantageously suitable to be conjugated with a wide range of functional molecules, such as chromophores, fluorophores and intercalating residues.
  • reaction schemes 1, 2, and 3 which illustrate the synthesis of the phosphoramidite of 2'-deoxyadenosine modified at position 8 with a bis-thioalkyl linker and conjugated with psoralen (scheme 1) or with acridine (scheme 2) and the synthesis of the phosphoramidite of 2'-deoxyadenosine modified at position 8 with a thioal- kynyl linker (scheme 3), respectively.
  • 3r-deoxyadenosine (1) is reacted with hexane-l,6-dithiol, to give the deoxyadenosine de- ivative modified at position 8, indicated with (2) in scheme 1.
  • the reaction takes place in in aqueous medium, preferably water and triethylamine (TEA preferably 10 eq.), at a temperature preferably ranging between 80-100° C, more preferably of about 100°C, and for a eaction time preferably ranging between 1.5 - 2.5 hours, more preferably of about 2 lours. rhe reaction proceeds with quantitative yields.
  • aqueous medium preferably water and triethylamine (TEA preferably 10 eq.
  • the derivative (2) can be selectively isolated by extraction with an organic solvent without chromatographic processes, which provides an important synthesis advantage.
  • a functional molecule such as, for example, psoralen, in the form of Br-methyl psoralen (3), to give the derivative (4).
  • the reaction takes place in an organic solvent, dry dimethylformamide, in the presence of calcium carbonate at a temperature preferably ranging between 60-80° C, more preferably of about 80°C, and for a reaction time preferably ranging between 2.5-3.15 hours, more preferably of about 3 hours.
  • rhe derivative (4) can be isolated as a solid product at the end of the work-up procedures, again without tedious and expensive chromatographic processes. rhe derivative (4) is then optionally transformed into the final product (7), which is a protected phosphoramidite, through a three-step process, the reaction conditions of which are described in detail in the following experimental section.
  • the three-step protection process provides firstly for the selective protection of the hydroxyl at the position 5' of the sugar radical with dimethoxytrityl chloride (DMT) to give the compound (5), which can be purified by chromatography.
  • DMT dimethoxytrityl chloride
  • the protection step of the ammino group at position 7 of the purine ring as the diphenylacetic acid amide to give the compound (6) follows, which compound can also be purified by chromatography.
  • the hydroxyl at position 3' of the sugar radical of the compound (6) is phosphorylated to give the compound (7), which can be purified by chromatography.
  • the first synthesis step is identical to that described before with reference to scheme 1, leading to the obtainment of the derivative (2) of scheme 2.
  • the terminal thiol group of the derivative (2) is easily conjugated with acridine, employed in the form of dichloromethoxy icridine (3), to give the derivative (4).
  • the reaction takes place in an organic solvent, dry dimethylformamide, in the p resence of triethylamine (TEA) at a temperature preferably ranging between 60-80° C, nore preferably of about 80°C, and for a reaction time preferably ranging between 15-17 lours, more preferably of about 17 hours.
  • rhe derivative (4) is easily isolated as a solid product at the end of the work-up procedures, without tedious and expensive chromatographic processes.
  • 3r-deoxyadenosine (1) is reacted with hex-5-yne-l -thiol, to give the deoxyadenosine de- ivative modified at position 8, indicated with (2a) in scheme 3.
  • the reaction takes place in in aqueous medium, preferably water and triethylamine (TEA), at a temperature preferably anging between 80-100° C, more preferably of about 100°C, and for a reaction time pref- ;rably ranging between 1.5-2.5, more preferably of about 2 hours. rhe reaction proceeds with quantitative yields.
  • aqueous medium preferably water and triethylamine (TEA)
  • TAA triethylamine
  • the derivative (2a) of scheme 3 can advantageously be isolated without chromatographic processes.
  • rhe reaction of scheme 3 is particularly advantageous, since it allows obtaining a deriva- ive suitable for use in click chemistry in very few passages that are easy to be carried out.
  • a derivative, having a terminal -C ⁇ CH can advantageously be conjugated with a nolecule containing an azide group, according to the click-chemistry methods.
  • molecules containing an azide group suitable to be conjugated with the derivative laving a terminal -C ⁇ CH are 6-carboxy-fluorescein azide, 5-carboxytetramethyl rhoda- nine azide, biotine azide, ferrocene-azide (for reference, see the web site vww.baseclick.eu).
  • the synthesis process of the present invention allows attaching a linker with a thiol bond iirectly to the position C8 of the adenosine, deoxyadenosine, 2'-0-alkyl adenosine or 2'- 3-silyl adenosine.
  • the specificity of this reaction is made possible by the synthesis in an iqueous environment, which ensures the proper solubility and reactivity of the reactants rnd allows the isolation of the product without the need of using expensive and tedious chromatographic methods.
  • This type of synthesis is specific for the adenine ring, since the iifference in the chemical potential of guanine, under similar reactions conditions, would ead to the formation of guanosine.
  • oligonucleotides were synthesized with a Gene Assembler II + automatic synthesizer, by using the original protocols, unless otherwise indicated.
  • df RP-18 special Pharmacia FPLC columns were used, with a gradient generated by a peristaltic pump operating at 1.2 mL/minute, collecting 8 mL fractions.
  • 8-Br-2'-deoxyadenosine was purchased from Berry & Associates, Dexeter, MI, U.S.A.
  • trimethoxy-psoralen was dissolved in 26 ml acetic acid (AcOH) and added with 1.89 mL bromomethyl ether (2.85 g, 22.8 mmol) and mixed at room temperature (RT). After 24 hours, a new aliquot of bromomethyl ether (2.85 g) was added, and the reaction was continued for additional 24 hours. The reaction mixture was then cooled in an ice bath for 2 hours, then filtered, and the solid obtained was washed with ethyl ether and concentrated in vacuum to yield 120 mg of the desired product. The stock solution, in combination with the washing fluid, was concentrated in vacuum and gave a new portion )f precipitate, which was washed and dried as before (136 mg). The two precipitated were assessed as having a comparable purity by NMR analysis, pooled, and subsequently used without further purifications. (80% yield).
  • rhe hydroxyl 5' protection product of the derivative described above was prepared by dis- iolving the latter (625 mg, 1 mmol) in 8 mL dry pyridine, adding fresh dimethoxytrityl :hloride (400 mg, 1.2 mmol). The reaction is carried out under stirring at RT in an inert itmosphere for 1 hour. The raw reaction raw product is concentrated to a small volume by vacuum evaporation, diluted in EtOAc (40 mL) and washed with 10% p/v citric acid in wa- er (4 x 10 mL) and finally, with water (2 x 10 mL).
  • the organic phase is purified on silica ;el by eluting with CH 2 Cl 2 /MeOH 98/2 v/v, thus obtaining the desired product in an 80% field.
  • the compound of the title was obtained by dissolving the dimethoxytritylated derivative of the conjugated adenosine-psoralen (928 mg 1.0 mmol) in 5 mL dry pyridine. To this, trimethylsilyl chloride (0.635 mL 5 mmol) is added in an inert atmosphere, the mixture is left under stirring at RT for 30 minutes. Diphenylacetyl chloride (227 mg 1.2 mmol) is then added, and it is left under stirring for further 2 hours.
  • the product derivative from the previous reaction (1.122 g 1.0 mmol) is dissolved in dry CH 2 C1 2 (5 mL) and added with ethyldiisopropylamine (0.85 mL, 5 mmol), cyanoethyl- diisopropyl chlorophosphite (0.36 mL, 1.6 mmol).
  • the reaction is kept stirred in an inert atmosphere at RT for 30 minutes.
  • the reaction is diluted with 30 mL CH 2 C1 2 and washed with NaHC0 3 (8% p/v in water) and subsequently with brine (10% p/v NaCl in water).
  • the product is purified by silica chromatography in a gradient of 20/80/10 ethyl ace- tate/cyclohexane/triethylamine and 90/10/10 yielding a spongy solid in a 50% yield.
  • 8-thiopentane-thioacridine-2'-deoxyadenosine (630 mg, 1.0 mmol) is dissolved in 8 mL dry pyridine and added with fresh dimethoxytrityl chloride (400 mg, 1.2 mmol).
  • the mixture is allowed to react for 1 hour at RT in an anhydrous atmosphere, after that it is concentrated to a small volume, re-dissolved in ethyl acetate (40 mL) and washed with 10% w/v citric acid in water (4 x 10 mL), and finally with water (2 x 10 mL).
  • the organic phase is purified on silica gel by eluting with CH 2 Cl 2 /MeOH/triethylamine 97/2/1 v/v, thus obtaining the desired product in a 65% yield.
  • the previous compound (1.120 g, 1 mmol) is dissolved in dry dichloromethane (5 mL) and ethyldiisopropylamine (0.85 mL, 5 mmol) and, finally, added with cyanoethyl-diisopropyl chlorophosphite (0.36 mL, 1.6 mmol).
  • the reaction is kept stirred in an inert atmosphere at RT for 30 minutes.
  • the reaction is diluted with 30 mL CH 2 C1 2 and washed with NaHC0 3 (8% p/v in water) and subsequently with brine (10% p/v NaCl in water).
  • the product is purified by chromatography on silica in a gradient of 20/80/10 and 90/10/10 ethyl ace- tate/cyclohexane/triethylamine, yielding a spongy solid in a 50% yield.
  • 6-chloro-l-hexine (0.60 mL, 5 mmol) is dissolved in dry DMF (5 mL). To the solution, potassium ethanethioate is added, then it is left at 50°C under stirring for 3 hours. The reaction mixture is extracted with Et 2 0 (2 x 15 mL) and washed with H 2 0 (5 x 30 mL). The solvent is brought to dryness in a rotavapor and the product is used without further purification (quantitative yield).
  • rhe compound obtained above (557 mg, 1.0 mmol) is dissolved in dry pyridine (5 mL) and • eacted, under a nitrogen atmosphere under stirring, with trimethylsilyl chloride (0.635 TiL, 5.0 mmol). After 30 minutes, the diphenylacetyl chloride (277 mg 1.2 mmol) is added, md the reaction is continued for 2 hours. MeOH (1 mL) is added, and after 15 minutes ⁇ 4 ⁇ (1 mL al 30% p/v). As in the corresponding psoralen derivative, the removal of :he silyl protecting group and the possible bis-protected amide is followed. The already described work-up is followed, with successive purification on silica gel with H 2 Cl 2 /MeOH/triethylamine 89/10/1, thus obtaining a 90% yield.
  • a solution is prepared, which contains the compound described above (557 mg, 1 mmol) in dry dichloromethane (5 mL) and ethyldiisopropylamine (0.85 mL, 5 mmol). To this solution, cyanoethyl-diisopropyl chlorophosphite (0.36 mL, 1.6 mmol) is added. The reaction is kept stirred in an inert atmosphere at RT for 30 minutes. The reaction is diluted with 30 mL CH 2 C1 2 and washed with NaHC0 3 (8% p/v in water) and subsequently with brine (10% p/v NaCl in water).
  • the product is purified by chromatography on silica in a gradient of 20/80/10 and 90/10/10 ethyl acetate/cyclohexane/triethylamine, yielding a spongy solid in a 60% yield.
  • amidites corresponding to the three conjugates are diluted in acetonitrile at a 0.1 mM concentration (0.08 mM in the case of the slightly less soluble acridine derivative), as any common phosphoramidite, and loaded on the Gene Assembler 11+ synthesizer in the position corresponding to that of the modified base X.
  • Syntheses of oligonucleotides having the following sequence: 5'CGTGCXTCCTAGC3' are carried out by using the standard protocols for the 1.3 ⁇ ⁇ synthesis scale, taking care of lengthening the coupling times by two minutes for the X base.
  • the standard coupling protocols (although with the times elongated by two minutes) provide a normal incorporation of the modified adenosines in the oligonucleotide synthesized with efficiency totally comparable, if not superior, to that of the standard amidite.
  • the cartridges are dried with an air jet and placed inside of a 1.6 mL plastic vial supplied with a watertight cap.
  • the cartridge is filled with 30% w/v aqueous ammonia, closed and left overnight (16 hours) at 50 °C to allow the release from the CPG and the removal of the protecting groups.
  • the cartridge is cooled and opened, and the liquid is transferred into a 12 mL tube.
  • the cartridge is washed thrice with 1 mL mil-li-Q water and the aqueous phases are combined to the previous ammonia solution.
  • TEA triethylammonium acetate
  • PI -norm 68.8 OD, equal to 0.59 ⁇ with a 36% whole yield in relation to the used CPG;
  • Pl-pso 64.15 OD, equal to 0.55 ⁇ with a 28% whole yield in relation to the used CPG
  • Pl-acr 63.32 OD, equal to 0.54 ⁇ with a 31% whole yield in relation to the used CPG
  • Pl-yne 104.64 OD, equal to 0.89 ⁇ with a 45% whole yield in relation to the used CPG.
  • the UV absorption spectra of the four oligonucleotides synthesized are those expected: ⁇ ⁇ 3 ⁇ 270 nm, Pl-pso shows a second absorption at 340 nm, Pl-acr shows a second absorption extending from 320 to 450 nm, while the absorption of the alkynyl group in Pl- yne is not measured in the visible range.
  • the purified oligonucleotides show the expected mass value by ESI spectrometry:

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  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne de nouveaux dérivés d'adénosine et de désoxyadénosine modifiés en position 8 du noyau purique, de formule générale (I), qui sont appropriés pour être conjugués avec des molécules fonctionnelles telles que des chromophores, des fluorophores et des résidus intercalants, et pour être incorporés dans des oligonucléotides synthétiques. L'invention concerne également un procédé de synthèse des dérivés susmentionnés qui permet de les obtenir à des rendements élevés et par des procédés simples. Formule (I) dans laquelle Z est sélectionné dans (a) et (b).
EP12731710.5A 2011-05-30 2012-05-29 Dérivés d'adénosine ou de désoxyadénosine modifiés en position 8 et leur procédé de synthèse Withdrawn EP2714708A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT000468A ITTO20110468A1 (it) 2011-05-30 2011-05-30 Derivati di adenosina o deossiadenosina modificati in posizione 8 e loro procedimento di sintesi
PCT/IB2012/052675 WO2012164484A1 (fr) 2011-05-30 2012-05-29 Dérivés d'adénosine ou de désoxyadénosine modifiés en position 8 et leur procédé de synthèse

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EP2714708A1 true EP2714708A1 (fr) 2014-04-09

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EP12731710.5A Withdrawn EP2714708A1 (fr) 2011-05-30 2012-05-29 Dérivés d'adénosine ou de désoxyadénosine modifiés en position 8 et leur procédé de synthèse

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EP (1) EP2714708A1 (fr)
IT (1) ITTO20110468A1 (fr)
WO (1) WO2012164484A1 (fr)

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CN109970832B (zh) * 2019-04-09 2022-06-03 沈阳药科大学 一种炔基修饰的脱氧腺苷亚磷酰胺单体及其制备方法
CN114685560B (zh) * 2020-12-31 2024-05-14 沈阳药科大学 含哌啶骨架亚磷酰胺单体及寡聚核苷酸的合成和应用

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WO2012164484A1 (fr) 2012-12-06
ITTO20110468A1 (it) 2012-12-01

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