Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/341—Gapmers, i.e. of the type ===---===
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• Oligonucleotides are short DNA or RNA oligomers that can be chemically synthesized for research and medical purposes. Oligonucleotides are typically prepared by a stepwise addition of nucleotide residues to produce a specific sequence.
• Current solid-phase synthesis manufacturing process of thiolated oligonucleotides uses repetition of four- reactions per cycle, namely deprotection (e.g. , detritylation), coupling, sulfurization, and capping.
• the capping step was designed to block any unreacted 5'-hydroxyl groups that failed to couple with the phosphoramidite in each cycle, so the failure sequence does not participate further in the coupling reactions in the subsequent cycles to generate impurities missing the nucleoside of the failed coupling.
• the capping step is done typically using acetic anhydride with base catalysts, such as pyridine or N-methyl morpholine.
• the invention herein describes a solid-phase synthesis manufacturing process for fully or partially thiolated oligonucleotides using repetition of a-three -reactions per cycle, namely detritylation, coupling, and sulfurization, rather than conventional four-reactions per cycle.
• nucleotides are added to a growing oligomer chain by coupling the free 5'- hydroxyl of the last nucleotide of the oligomer attached to the solid support with the appropriate phosphoramidite.
• Inefficiencies in the coupling reaction would leave some of the 5'-hydroxyl groups free to react in subsequent coupling reactions, resulting in a complicated mixture of oligomers missing a single, but random, nucleotide.
• the 5'-hydroxyl is capped via the use of a capping reagent such as acetic anhydride prior to or after the sulfurization step.
• the present inventors developed a process that eliminates the capping step prior to or following the sulfurization step.
• the present inventors have surprisingly discovered that the byproducts generated during the sulfurization step can be used to cap the unreacted 5'- hydroxy groups.
• the sulfurization agent itself for example PADS, may be recirculated across the solid support to cap the 5 '-hydroxy groups that failed to couple with the phosphoramidite.
• the unreacted 5' hydroxyl groups react with the sulfurization byproducts, without the need of a separate capping step, and are no longer available to couple in the subsequent cycles.
• no capping step is needed at all and the process includes only 3 reactions per cycle (deprotection, coupling, and sulfurization).
• Use of the process described herein can be used to eliminate the capping step following the sulfurization step. However, because the byproducts of the oxidation step do not cap the unreacted 5 '-hydroxy, residual amounts of unreacted 5 '-hydroxyl groups may remain following the oxidation step.
• the process described here in can be used even without a capping step following the oxidation step.
• the process described herein can be used but a conventional capping step, for example, with acetic anhydride, may be used following each oxidation step.
• FIG. 1 is a diagram of the 3-step oligonucleotide synthetic method.
• FIG. 2 is a diagram of the conventional 4-step oligonucleotide synthetic method. It is noted that the sulfurization step may be performed before or after the capping step in the conventional 4-reactions per cycle process.
• FIG. 3 is a chromatogram comparing the purity by mass spectrometry for the 4- reactions-cycle with PADS (top); 3 -reactions-cycle with PADS (middle); and 3-reactions- cycle with xanthane hydride (bottom).
• FIGs. 4 and 5 are RPIP HPLC chromatograms comparing the BIIB078 prepared by the 4-reactions-cycle process (bottom) and the 3 -reactions-cycle process (top) with PADS as the sulfurization agent.
• FIGs. 6 and 7 are mass spectrometry chromatograms comparing BIIB078 prepared by the 4-reactions-cycle process (bottom) and the 3 -reactions-cycle process (top) with PADS as the sulfurization agent.
• FIG. 8 shows 1H NMR spectra for reaction mixture of BuOH, P(OMe) 3 and PADS over time, wherein PADS was aged with 3-picoline in acetonitrile- ⁇ i?.
• FIG. 9 shows 1H NMR spectra for reaction mixture of BuOH, P(OMe) 3 and PADS over time, wherein PADS was aged with N-methyl imidazole in acetonitrile- ⁇ i?.
• Each R 1 and R 11 are independently a nucleobase, wherein the NH 2 of the nucleobase, if present, is protected by an amine protecting group;
• Each R 2 and R 12 are independently selected from the group consisting of H, halo, and Ci_ 6 alkoxy optionally substituted with Ci_ 6 alkoxy;
• R is a solid support, optionally comprising a linker
• Y is absent, a nucleotide, or an oligonucleotide comprising 2 or more nucleotides
• Each R 4 is independently H or forms a ring with the alkoxy group of R 2 ;
• Each R 14 is independently H or forms a ring with the alkoxy group of R 12 ;
• R 15 is a hydroxy protecting group
• R 16 is Ci_ 6 alkyl optionally substituted with -CN;
• R 17a and R 17b are independently Ci_ 6 alkyl
• R is a hydroxy protecting group formed from a byproduct of the sulfurization agent; wherein the sulfurization agent is reacted for a sufficient amount of time to substantially completely convert unreacted compound of Formula (I) to the compound of Formula (V) and to convert the compound of Formula (III) to Formula (IV);
• a sufficient amount of time could be determined by one of skill in the art based upon the reaction conditions. For example, the times provided in the thirteenth embodiment, namely 0 to 30 minutes, for example, 0, 5, 10, 15, 20, 25, or 30 minutes, would be “a sufficient amount of time”.
• substantially completely convert could be determined by one of skill in the art based upon the reaction conditions. In particular, substantially completely convert means to convert 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 % or 100 % of the unreacted compound of Formula (I) to Formula (V) or Formula (III) to Formula (IV).
• the process does not include an additional capping step following step b. Specifically, any unreacted compound of Formula (I), (VI), or (IX) is capped by the byproducts of the sulfurization step b without the require add any other reagent to cap any unreacted compound of Formula (I), (VI), or (IX) .
• R 12 is selected from the group consisting of H, halo, Ci_ 4 alkoxy, and Ci_ 4 alkoxyCi_ 4 alkoxy;
• Each R 14 is independently H or forms a 5- or 6-membered ring with the alkoxy group of R 12 ;
• R 15 is a hydroxy protecting group selected from 4,4'-dimethoxytrityl, [bis-(4- methoxyphenyl)phenylmethyl] (DMT) or trityl (triphenylmethyl, Tr).
• R 16 is Ci_ 4 alkyl substituted with -CN.
• R 17a and R 17b are independently Ci_ 4 alkyl.
• Formula (II) is selected from any one of the following structural formula:
• DMTO is the 4,4'-dimethoxytrityl protected 5 '-hydroxy and R 11 is as for the first embodiment.
• a second embodiment of the invention is a process as described for the first embodiment or any aspect thereof, further comprising the step of c) deprotecting the compound of Formula (IV) to form the compound of Formula (VI):
• a third embodiment of the invention is a process as described for the second embodiment, further comprising the step of d) reacting the compound of Formula (VI) with a compound of Formula (II) to form a compound of Formula (VII):
• a fourth embodiment of the invention is a process as described for the third embodiment, further comprising the step of f) deprotecting the compound of Formula (VIII) to form the compound of Formula (IX):
• a fifth embodiment of the invention is a process as described for the second and fourth embodiments, wherein starting with the compound of Formula (VI) or the compound of Formula (IX), steps a), b), and c) are repeated n times and steps d), e), and f) are repeated m times, wherein repetition of steps a), b), and c) and steps d), e) and f) can occur in any order relative to each other, wherein n is at least 1 or more and m is 0, 1, 2, 3, or 4, to form an oligonucleotide of Formula (X) or (XI):
• each X is independently selected from S or O, provided that X is O only 4, 3, 2, 1, or 0 times in the compound of Formula (X), (XI), (XII), and (XIII).
• n is 1 to 30, 5 to 25, or 10 to 15 and m is 0, 1, 2, 3, or 4.
• the process described above for the fifth embodiment involves a stepwise addition of nucleotides to form the desired oligonucleotide. Each of these steps involve three reactions to add the nucleotide to the growing oligonucleotide.
• the first step corresponding to steps a and d
• the 5' end of a nucleoside such as a compound of Formula (I)
• the 3' end of nucleotide such as a compound of Formula (II)
• the oligonucleotide is either sulfurized (step b) or oxidized (step e), respectively.
• step c or f the next 5 '-hydroxy group is deprotected (steps c or f, and for example, Formulas (VI) and (IX)) and the process is repeated to add the next nucleoside.
• this process allows for the synthesis of an oligonucleotide with any number of phosphothioate bonds and up to four phosphodiester without the need for any capping step at all because any unreacted 5 '-hydroxy groups will react with the sulfurization agent itself or a byproduct of the reaction of the sulfurization agent with the phosphorous atom in the linkage between two nucleosides.
• This is in contrast to the conventional synthesis, where a capping step to block any of the unreacted 5' OH is done following the coupling step.
• a “capping agent” may be any capping agent known to one of skill in the art, for example, acetic anhydride with base catalysts, such as pyridine or N-methyl morpholine.
• a seventh embodiment of the invention is a process as described for the sixth embodiment, further comprising the step of h) deprotecting the compound of Formula (VIII) to form the compound of Formula (XV):
• a eighth embodiment of the invention is a process as described for the seventh embodiment, wherein starting with the compound of Formula (VI) or the compound of Formula (IX), steps a), b), and c) are repeated n times and steps d), e), g) and h) are repeated m times, wherein repetition of steps a), b), and c) and steps d), e), g) and h) can occur in any order relative to each other, wherein n is at least 1 and m is 0 or greater to form an oligonucleotide of Formula (X) or (XVII):
• XIII wherein each X is independently selected from S or O.
• n is 1 to 30, 5 to 25, or 10 to 15 and m is 0 to 10, 0 to 5, or 5 to 10.
• the process described above for the eighth embodiment involves a stepwise addition of nucleotides to form the desired oligonucleotide. Each of these steps involve three reactions to add a nucleotide containing a phosphothioate linkage to the growing
• oligonucleotide and four reactions to add a nucleotide containing a phosphodiester linkage to the growing oligonucleotide.
• the 5' end of a nucleoside (such as a compound of Formula I) is coupled to the 3' end of nucleotide (such as a compound of Formula II) to couple these two nucleotides (forming compounds such as those of Formula (III) and (VII).
• the oligonucleotide is either sulfurized (step b, forming compound such as those of Formula (IV)) or oxidized (step e, forming compound such as those of Formula (IV) or (VIII)).
• step g forming compound such as those of Formula (IV)
• step g no capping step is required following the sulfurization step.
• next 5 '-hydroxy group is deprotected (steps c or h, and for example, Formulas (VI) and (IX)) and the process is repeated to add the next nucleoside. Therefore, this process allows for the synthesis of an oligonucleotide with any number of phosphothioate bonds and phosphodiester bond, and requires a capping step only after the cycle including an oxidation step. This is in contrast to the conventional synthesis, where a capping step to block any of the unreacted 5 ⁇ is done every cycle, even those including a sulfurization step.
• the compounds of Formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), or (XV) are not salts.
• n is 2 to 1000. In particular, n is 2 to 500. In particular, n is 2 to 50. In particular, n is 2 to 25.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, and eighth embodiments, or any aspect thereof, wherein the linker attached to the solid support is cleaved.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth embodiments, or any aspect thereof, the sulfurization agent is a compound of Formula A: R 1 ,
• R 1 and R 2 are both H:
• R 1 and R 2 are both methyl:
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth embodiments, or any aspect thereof, the sulfurization agent is a compound of Formula B:
• each of R 1 and R 2 are independently selected from H, an optionally substituted alkyl and an optionally substituted aryl group.
• R and R are both benzyl (PhCH 2 ):
• the sulfurization agent is 3H-l,2-benzodithiol-3-one 1,1-dioxide (Beaucage Reagent):
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, and twelfth embodiments, or any aspect thereof, wherein the sulfurization agent is reacted by
• the sulfurization agent is recirculated for 0 to 30 minutes, for example, 0, 5, 10, 15, 20, 25, or 30 minutes. In one embodiment, the sulfurization agent is recirculated for 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, or 1 to 5 minutes. In another embodiment, the sulfurization agent is recirculated for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth embodiments, or any aspect thereof, wherein the sulfurization agent is recirculated at least 0-20 times, for example, 1, 2, 5, 10, 15, 20 times.
• the sulfurization agent is recirculated at least 0-20 times, for example, 1, 2, 5, 10, 15, 20 times.
• sulfurization agent is recirculated for 1-20 times, 1-10 times, or 1-5 times.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, and fourteenth embodiments, or any aspect thereof, the sulfurization agent is reacted by recirculating 3 to 6 equivalents of sulfurization agent relative to the amount of the first nucleoside attached to the solid support.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and fifteenth embodiments, or any aspect thereof, the concentration of the sulfurization reagent is from 0.02 M to 2.0 M.
• the sulfurization agent is dissolved in pyridine, 3-piccoline, acetonitrile, or a mixture thereof.
• the concentration of the sulfurization reagent is from 0.05 M to 0.5 M.
• the sulfurization reagent is 3-(dimethylamino-methylidene)amino)-3H- 1,2,4-dithiazole (DDTT) added at a concentration of 0.02 M to 0.1 M, for example, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, or 0.1M dissolved in pyridine.
• DDTT 3-(dimethylamino-methylidene)amino)-3H- 1,2,4-dithiazole
• the sulfurization reagent is 3H-l,2-benzodithiol-3-one 1,1-dioxide (Beaucage Reagent) added at a concentration of 0.01 M to 0.2 M, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 M in acetonitrile (ACN).
• ACN acetonitrile
• the sulfurization reagent is phenylacetyl disulfide (PADS) added at a concentration of 0.01 M to 0.2 M, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 M in acetonitrile (ACN)/3-picoline.
• PADS phenylacetyl disulfide
• the sulfurization reagent is 3-amino- 1,2,4- dithiazole-5-thione (XH or ADTT) added at a concentration of 0.01 M to 0.2 M, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 M in pyridine.
• XH or ADTT 3-amino- 1,2,4- dithiazole-5-thione
• the sulfurization reagent described above e.g. , phenylacetyl disulfide (PADS) or 3-amino- l,2,4-dithiazole-5-thione (XH)
• a base i.e. , aging
• Exemplary base includes, but is not limited to, N-methylimidazole, pyridine and 3-picoline.
• the sulfurization reagent can be aged with the base (i.e. , treated with the base) for 1 minute to 5 days, for example for 5 minutes to 24 hours, 10 minutes to 12 hours, 30 minutes to 10 hours, 1 hour to 8 hours, 2 hours to 6 hours, or 3 hours to 5 hours.
• the sulfurization reagent is aged for 4 hours.
• the sufurization reagent PADS is aged with 3-picoline for 4 hours before being used in the process of the present invention.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, and sixteenth embodiments, or any aspect thereof,
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, and seventeenth embodiments, or any aspect thereof, each R
• R are independently selected from the group consisting of H, F, and Ci_ 4 alkoxy optionally substituted with Ci_ 4 alkoxy; each R 4 is independently H or forms a ring with the alkoxy group of R , wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 CM alkyl groups; R 16 is -CH 2 CH 2 CN; R 17a and R 17b are independently CM alkyl; and each R 14 is independently H or forms a ring with the alkoxy group of R 12 , wherein the ring is a 5 or 6-membered ring optionally substituted with 1 to 3 Ci_ 4 alkyl groups.
• each R 2 and R 12 are independently selected from H or Ci_ 4 alkoxy optionally substituted with Ci_ 4 alkoxy;
• R 15 is 4,4'-dimethoxytrityl;
• R 16 is -CH 2 CH 2 CN;
• R 17a and R 17b are independently Ci_ 6 alkyl; and
• Y is absent.
• Y is a single nucleotide.
• Y is
• oligonucleotide comprising 2 to 50 nucleotides, in particular, 2 to 40 nucleotides, 2 to 30 nucleotides, or 2 to 25 nucleotides.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth
• the compound of Formula (X) or (XI) is an anti-sense oligonucleotide comprising 2 to 30 nucleotides.
• the anti-sense oligonucleotide comprises modified RNA only.
• the anti-sense oligonucleotide comprises DNA and modified RNA.
• the anti-sense oligonucleotide is a gapmer.
• the anti-sense oligonucleotide comprises DNA only.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty- first embodiments, or any aspect thereof,
• the anti-sense oligonucleotide is a phosphorothioate oligonucleotide having a sequence of (from 5' to 3')
• TCACTTTCATAATGCTGG (SEQ ID NO: 1), wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage, each nucleoside of the oligonucleotide is a 2'-0- methoxyethyl (MOE) nucleoside, and each cytosine is a 5-methylcytosine.
• SEQ ID NO: 1 is also known as BIIB058, and is described in WO2007/002390, WO2010/148249, and US8,980,853, the teaching of each are herein incorporated by reference.
• the process is as described for any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty- first embodiments, or any aspect thereof, the sequence of the anti-sense oligonucleotide is a 5-10-5 MOE gapmer, having a sequence of (from 5' to 3')
• CAGGATACATTTCTACAGCT SEQ ID NO: 2
• each of nucleosides 1-5 and 16-20 are 2'-0-methoxyethylribose modified nucleosides
• each of nucleosides 6- 15 are 2'-deoxynucleosides
• internucleoside linkages between nucleosides 2 to 3, 4 to 5, 16 to 17, and 18 to 19 are phosphodiester linkages and the internucleoside linkages between nucleosides 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 17 to 18, and 19 to 20 are phosphorothioate linkages, and wherein each cytosine is a 5'- methylcytosine.
• SEQ ID NO:2 is described by the following chemical notation: mCes Aeo Ges Geo Aes Tds Ads mCds Ads Tds Tds Tds mCds Tds Ads mCeo Aes Geo mCes Te; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• o a phosphodiester internucleoside linkage.
• SEQ ID NO: 2 is as known as BIIB067 or ISIS 666853 and is described in
• the anti-sense oligonucleotide is a 4-8-6 gapmer, having a sequence of (from 5' to 3'):
• GCCCCTAGCGCGCGACUC (SEQ ID NO: 8) wherein each of nucleosides 1-4 and 13-18 are 2'-0-methoxyethylribose modified nucleosides, and each of nucleosides 5-12 are 2'-deoxy ribonucleotides, wherein the intemucleoside linkages between nucleosides 2 to 3, 3 to 4, 13 to 14, 14 to 15, and 15 to 16 are phosphodiester linkages and the intemucleoside linkages between nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 16 to 17 and 17 to 18 are phosphorothioate linkages, wherein each cytosine is 5-methylcytosine, and wherein the uracil is 5-methyluracil.
• SEQ ID NO:8 is described by the following chemical notation:
• Me U 5-methyluracil (also known as thymine)
• the 5 '-hydroxy that failed to couple during the coupling step forms a hydroxyl derivative from sulfurization with the sulfurization agent, such as, for example, PADS, or a N-cyanocarbonimidothioate from xanthane hydride, or N,N-dimethylthiourea-N-thiocarboxylate from DDTT:
• the sulfurization agent such as, for example, PADS, or a N-cyanocarbonimidothioate from xanthane hydride, or N,N-dimethylthiourea-N-thiocarboxylate from DDTT:
• failure sequence capped failure sequence A "hydroxy protecting group” includes any group suitable for protecting a hydroxy group, OH. These protecting groups and further examples of hydroy protecting groups can be found in Greene, TW et al., Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons (2007).
• the hydroxy protecting group is formed from the capping agent ⁇ e.g., R c in Formula (XIV)), and can be selected from, for example, acetyl (Ac);
• TMS trimethylsilyl
• TDMS tert-butyldimethylsilyl
• TOM tri
• the hydroxy protecting group blocks a 5'-hydoxy of a nucleoside or nucleotide ⁇ e.g., R 15 in Formulas (II), (III), (IV), (VII), and (VIII)), and in particular aspect is an acid-labile protecting group selected from 4,4'-dimethoxytrityl, [bis-(4- methoxyphenyl)phenylmethyl] (DMT) or trityl (triphenylmethyl, Tr).
• R 15 in Formulas (II), (III), (IV), (VII), and (VIII)
• an acid-labile protecting group selected from 4,4'-dimethoxytrityl, [bis-(4- methoxyphenyl)phenylmethyl] (DMT) or trityl (triphenylmethyl, Tr).
• Nucleobase means the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or may be modified. In certain embodiments, a nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a nucleobase of another nucleic acid. In particular, the nucleobase is a heterocyclic base, typically purines and pyrimidines.
• a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp.
• nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
• Nucleoside means a compound comprising a heterocyclic base moiety and a sugar moiety.
• Nucleotide means a nucleoside comprising a phosphate linking group.
• Oligonucleotide refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
• Internucleoside linkage means a covalent linkage between adjacent nucleosides of an oligonucleotide.
• Gapmer means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
• alkyl refers to a fully saturated branched or unbranched hydrocarbon moiety.
• the alkyl comprises 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
• an alkyl comprises from 6 to 20 carbon atoms.
• alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.
• aryl refers to monocyclic, bicyclic or tricyclic aromatic hydrocarbon groups having from 6 to 14 carbon atoms in the ring portion. In one embodiment, the term aryl refers to monocyclic and bicyclic aromatic hydrocarbon groups having from 6 to 10 carbon atoms. Representative examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl.
• aryl also refers to a bicyclic or tricyclic group in which at least one ring is aromatic and is fused to one or two non-aromatic hydrocarbon ring(s).
• Nonlimiting examples include tetrahydronaphthalene, dihydronaphthalenyl and indanyl.
• Optional substituents for both the alkyl or aryl groups are, in each occurrence, independently selected from Ci_ 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, 3- to 7-membered
• R a in each occurrence is independently selected from H, Ci_ 6 alkyl, 3- to 6-membered monocyclic carbocyclyl, and 3- to 6-membered monocyclic heterocyclyl.
• carbocyclyl refers to saturated or unsaturated monocyclic or bicyclic hydrocarbon groups of 3-7 carbon atoms, 3-6, or 5-7 carbon atoms.
• carbocyclyl encompasses cycloalkyl groups and aromatic groups.
• cycloalkyl refers to completely saturated monocyclic or bicyclic hydrocarbon groups of 3-7 carbon atoms, 3-6 carbon atoms, or 5-7 carbon atoms.
• Exemplary monocyclic carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopropenyl, cyclobutenyl, cyclopenentyl, cyclohexenyl, cycloheptenyl, cyclobutadienyl, cyclopentadienyl, cyclohexadienyl, cycloheptadienyl, phenyl and cycloheptatrienyl.
• Exemplary bicyclic carbocyclyl groups include bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, tricyclo[2.2.1.0 2 ' 6 ]heptanyl, 6,6-dimethylbicyclo[3.1.1]heptyl, or 2,6,6-trimethylbicyclo[3.1.1]heptyl, spiro[2.2]pentanyl, and spiro[3.3]heptanyl.
• bridged ring system is a ring system that has a carbocyclyl or heterocyclyl ring wherein two non-adjacent atoms of the ring are connected (bridged) by one or more (preferably from one to three) atoms selected from C, N, O, or S.
• a bridged ring system may have from 6-7 ring members.
• spiro ring system is a ring system that has two rings each of which are independently selected from a carbocyclyl or a heterocyclyl, wherein the two ring structures having one ring atom in common. Spiro ring systems have from 5 to 7 ring members.
• heterocyclyl refers to a saturated or unsaturated, monocyclic or bicyclic (e.g., bridged or spiro ring systems) ring system which has from 3- to 7-ring members, or in particular 3- to 6- ring members or 5- to 7- ring members, at least one of which is a heteroatom, and up to 4 (e.g., 1, 2, 3, or 4) of which may be heteroatoms, wherein the heteroatoms are independently selected from O, S and N, and wherein C can be oxidized (e.g., C(O)), N can be oxidized (e.g., N(O)) or quaternized, and S can be optionally oxidized to sulfoxide and sulfone.
• C can be oxidized
• N can be oxidized
• S can be optionally oxidized to sulfoxide and sulfone.
• heteroaryl refers to an aromatic 5 or 6 membered monocyclic ring system, having 1 to 4 heteroatoms independently selected from O, S and N, and wherein N can be oxidized (e.g., N(O)) or quaternized, and S can be optionally oxidized to sulfoxide and sulfone.
• a heterocyclyl is a 3-to 7-membered saturated monocyclic or a 3-to 6-membered saturated monocyclic or a 5-to 7-membered saturated monocyclic ring.
• a heterocyclyl is a 3-to 7-membered monocyclic or a 3-to 6-membered monocyclic or a 5-to 7-membered monocyclic ring.
• a heterocyclyl is a 6 or-7-membered bicyclic ring.
• the heterocyclyl group can be attached at a heteroatom or a carbon atom.
• heterocyclyls include aziridinyl, oxiranyl, thiiranyl, oxaziridinyl, dioxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, thiolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, oxathiolanyl, piperidinyl, tetrahydropyranyl, thianyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, dithianyl, trioxanyl, trithianyl, azepanyl, oxepanyl, thiepanyl, dihydro
• bicyclic heterocyclic ring systems include 3-azabicyclo[3.1.0]hexanyl, 3- azabicyclo[3.1.1]heptanyl, 2-azaspiro[3.3]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, and 5- azaspiro[2.3]hexanyl.
• Halogen or “halo” may be fluoro, chloro, bromo or iodo.
• the coupling step was carried out by circulating a solution of corresponding phosphor amidite (0.1- 0.2 M in acetonitrile) and 4,5- dicyanoimidazole (1.0 M in acetonitrile containing 0.1M N-methylimidazole) through the solid support.
• the sulfurization step was completed by circulating any one of the following solutions: (a) 0.2 M PADS in acetonitrile/3-picoline (1: 1, v/v); (b) 0.2 M 3 -Amino- 1,2,4- dithiazole-5-thione (XH) in Pyridine; (c) 0.2 M 3H-l,2-Benzodithiol-3-one 1,1- Dioxide (Beaucage Reagent) in Acetonitrile; or (d) 0.1 M 3-((Dimethylamino- methylidene)amino)-3H-l,2,4-dithiazole-3-thione (DDTT) in Pyridine.
• the synthesis consists of four reaction steps: detritylation, coupling, and sulfurization, and capping step. All the steps are carried out the same way as above, except with a capping step after the sulfurization step.
• the capping was done with a mixture of acetic anhydride, pyridine, and N-methyl imidazole in acetonitrile. Cleavage of the oligonucleotides from the support and base deprotection were performed in concentrated ammonium hydroxide at elevated temperature (50 to 60 °C).
• the synthesis reaction parameters are summarized in Table 1 below. The oligonucleotide products were analyzed by LC-MS.
• BIIB 058 SEQ ID NO: 1
• each riboligonucleotide includes methoxy-ethyl (MoE) at the 2' position.
• BIIB 067 (SEQ ID NO: 2), a 5-10-5 gapmer phosphothioester and phosphodiester mixed backbone oligonucleotide.
• the central block of a gapmer is 10 deoxy ribonucleotides, which is flanked by blocks of 2' -MoE ribonucleotides.
• BIIB 078 (SEQ ID NO:8), a 4-8-6 gapmer phosphothioester and phosphodiester mixed backbone oligonucleotide.
• the central block of a gapmer is 8 deoxy ribonucleotides, which is flanked by blocks of 2' -MoE ribonucleotides.
• BIIB058 was prepared using the process of the invention (i.e., the 3 -reactions-cycle) and compared to the conventional 4-reactions per cycle process.
• PADS and Xanthane Hydride were used as the sulfurization agent.
• the sulfurization reaction was in recirculation mode for 5 min. The results of each process are shown in Table 3.
• BIIB067 was prepared using the process of the invention (i.e., the 3-reactions-cycle) and compared to the conventional 4-reactions per cycle process, with PADS used as sulfurization agent.
• the sulfurization reaction was in recirculation mode for 5 min.
• Table 4 The results of each process are shown in Table 4.
• Oligonucleotides A, B, C, D, and E were prepared using the process of the invention and compared to the conventional 4-reactions per cycle process.
• Table 5 provides the amidites used for the synthesis.
• the sulfurization agents used are shown in Table 6.
• the yield, UV purity, and MS impurity profile of crude oligonucleotide from the process of the invention is similar to the crude oligonucleotide as prepared by the conventional 4-reactions-cycle process.
• MS impurity profile from 3 -Reactions- cycle process is compatible as the crude from 4- Reactions-cycle process.
• BIIB078 was prepared using the process of the invention (i.e., the 3-reactions-cycle) and compared to the conventional 4-reactions per cycle process.
• PADS was used as the sulfurization agent.
• the sulfurization reaction was in recirculation mode for 3 min. The results of each process are shown in Table 8 and FIGs. 4-7.

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