WO2004055030A1 - Process for the preparation of phosphitylation agents - Google Patents
Process for the preparation of phosphitylation agents Download PDFInfo
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- WO2004055030A1 WO2004055030A1 PCT/GB2003/005473 GB0305473W WO2004055030A1 WO 2004055030 A1 WO2004055030 A1 WO 2004055030A1 GB 0305473 W GB0305473 W GB 0305473W WO 2004055030 A1 WO2004055030 A1 WO 2004055030A1
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- WO
- WIPO (PCT)
- Prior art keywords
- formula
- compound
- process according
- group
- toluene
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000005731 phosphitylation reaction Methods 0.000 title description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Natural products CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 54
- 150000001875 compounds Chemical class 0.000 claims abstract description 44
- 239000002904 solvent Substances 0.000 claims abstract description 18
- 239000004215 Carbon black (E152) Substances 0.000 claims abstract description 7
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical group [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229930195733 hydrocarbon Natural products 0.000 claims abstract description 7
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 6
- 150000002430 hydrocarbons Chemical class 0.000 claims abstract description 6
- 229910052736 halogen Inorganic materials 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 4
- 125000003944 tolyl group Chemical group 0.000 claims abstract 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- -1 2-nitrophenyl Chemical group 0.000 claims description 8
- 150000001412 amines Chemical class 0.000 claims description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 125000004182 2-chlorophenyl group Chemical group [H]C1=C([H])C(Cl)=C(*)C([H])=C1[H] 0.000 claims description 2
- 125000003854 p-chlorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1Cl 0.000 claims description 2
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims 2
- 125000001309 chloro group Chemical group Cl* 0.000 claims 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims 1
- 125000001183 hydrocarbyl group Chemical group 0.000 claims 1
- 150000002367 halogens Chemical group 0.000 abstract description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 108091034117 Oligonucleotide Proteins 0.000 description 8
- 238000004821 distillation Methods 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000010992 reflux Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 150000007530 organic bases Chemical class 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 2
- CSDQQAQKBAQLLE-UHFFFAOYSA-N 4-(4-chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine Chemical compound C1=CC(Cl)=CC=C1C1C(C=CS2)=C2CCN1 CSDQQAQKBAQLLE-UHFFFAOYSA-N 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 2
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 229940124598 therapeutic candidate Drugs 0.000 description 2
- RFJDCAMETMDBBK-UHFFFAOYSA-N N-[2-benzhydrylsilylethoxy-[di(propan-2-yl)amino]phosphanyl]-N-propan-2-ylpropan-2-amine Chemical compound C=1C=CC=CC=1C([SiH2]CCOP(N(C(C)C)C(C)C)N(C(C)C)C(C)C)C1=CC=CC=C1 RFJDCAMETMDBBK-UHFFFAOYSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- YKLJUGWFWPZZFA-UHFFFAOYSA-N amino-(morpholin-4-ylamino)phosphinous acid Chemical compound NP(O)NN1CCOCC1 YKLJUGWFWPZZFA-UHFFFAOYSA-N 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- URAZVWXGWMBUGJ-UHFFFAOYSA-N di(propan-2-yl)azanium;chloride Chemical compound [Cl-].CC(C)[NH2+]C(C)C URAZVWXGWMBUGJ-UHFFFAOYSA-N 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000011363 dried mixture Substances 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- AUHZEENZYGFFBQ-UHFFFAOYSA-N mesitylene Substances CC1=CC(C)=CC(C)=C1 AUHZEENZYGFFBQ-UHFFFAOYSA-N 0.000 description 1
- 125000001827 mesitylenyl group Chemical group [H]C1=C(C(*)=C(C([H])=C1C([H])([H])[H])C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- FAIAAWCVCHQXDN-UHFFFAOYSA-N phosphorus trichloride Chemical compound ClP(Cl)Cl FAIAAWCVCHQXDN-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/26—Amides of acids of phosphorus containing P-halide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
-
- 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
- the present invention concerns a process for the preparation of phosphitylation agents.
- Synthetic oligonucleotides are important diagnostic tools for the detection of genetic and viral diseases.
- oligonucleotides and modified oligonucleotides are of interest as therapeutic candidates that inhibit gene expression or protein function.
- Large scale synthesis of oligonucleotides for use as therapeutic candidates has become increasingly important since FDA approval of an oligonucleotide analog for the treatment of cytomegalovirus (CMV), and several other oligonucleotide analogs are currently in clinical trials. Kilogram quantities of a purified oligonucleotide analog are needed for each clinical trial.
- CMV cytomegalovirus
- oligonucleotide The principal method currently employed for the preparation of oligonucleotide is the phosphoramidite approach.
- the increasing demand for larger quantities of oligonucleotides has correspondingly increased demand for phosphoramidite compounds.
- Phosphoramidite compounds are commonly prepared by phosphitylation of a nucleoside with a phosphitylation agent in the presence of an activator. Accordingly, there has been a corresponding increase in demand for phosphitylation agents. Improved or alternative processes for the preparation of phosphitylation agents are therefore needed.
- a process for the preparation of a compound of formula R 1 -Y 1 -P(NR 2 R 3 ) 2 which comprises: a) reacting a compound of formula PX 3 with a compound of formula HNR 2 R 3 to form a compound of formula X-P(NR 2 R 3 ) 2 ; and b) reacting the compound of formula X-P(NR 2 R 3 ) 2 with a compound of formula R 1 -Y 1 -H in the presence of a solvent to form the compound of formula R 1 -Y 1 -P(NR 2 R 3 ) 2 wherein
- R 1 represents a phosphorus protecting group
- R 2 and R 3 each independently represent an alkyl, preferably a C ⁇ alkyl, group, or R 2 and
- R 3 are joined, together with the N to which they are attached, to form a 5-7 membered ring;
- Y 1 represents O or S, preferably O;
- X represents a halogen, preferably Cl; characterised in that the solvent employed in reaction b) is a hydrocarbon solvent.
- Phosphorus protecting groups represented by R 1 are commonly cleavable phosphorus protecting groups employed in oligonucleotide synthesis, for example substituted or unsubstituted aliphatic groups, such as a methyl group, -CH 2 CH 2 -
- R 1 -Y 1 -H are preferably selected based on the nature of the compound it is desired to produce.
- An especially preferred compound of formula R 1 -Y 1 -H is 2-cyanoethanol.
- R 2 and R 3 are the same. It is particularly preferred that both R 2 and R 3 are -CH(CH 3 ) 2 groups. It is especially preferred that Y 1 is O and R 1 is -CH 2 CH 2 CN.
- Examples of compounds which can be prepared by the process of the present invention include O- ⁇ -cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, (commonly known as "tetraphos"), O- ⁇ -cyanoethyl-N,N,N',N'-tetramethylphosphorodiamidite, O- ⁇ - cyanoethyl-N,N,N',N'-tetraethylphosphorodiamidite, bis (N,N-diisopropylamino)-2- methyltrifluoroacetyiamino-ethoxyphosphine, bis (N,N-diisopropylamino)-2- diphenylmethylsilylethoxyphosphine and O- ⁇ -cyanoethyl-bis (N-morpholino) phosphorodiamidite.
- tetraphos O- ⁇ -cyanoethyl-N,N,N',N
- Hydrocarbon solvents that can be employed in the process of the present invention include aliphatic and aromatic hydrocarbons.
- aliphatic hydrocarbons include pentane, hexane and petroleum ethers.
- aromatic hydrocarbons include benzene, toluene, xylene and mesitylene. Toluene is the most preferred solvent.
- reaction a) takes place in the presence of the same solvent as reaction b), and most preferably in the presence of toluene.
- Bases which can be employed include inorganic bases, such as sodium carbonate, and organic bases.
- Organic bases are preferred. Examples of organic bases include aromatic amines such as pyridine, and inorganic amines, such as alkylamines, preferably trialkylamines, such as tr C- M alkyOamines, and most preferably triethylamine.
- the mole ratio of compound of formula PX 3 to compound of formula HNR 2 R 3 in step a) is commonly selected to be in the range of from about 1 : 1 to about 10 : 1 , and preferably from about 3 : 1 to about 6 : 1.
- the mole ratio of compound of formula X-P(NR 2 R 3 ) 2 to compound of formula R 1 - Y 1 -H in step b) is commonly selected to be in the range of from about 1 : 1 to about 5 : 1 , with mole ratios in the range of from 1 : 1 to 1.5 : 1 being especially preferred.
- X-P(NR 2 R 3 ) 2 is often in the range of from about 0.75 : 1 to 2 : 1 , and preferably from about 1 : 1 to 1.3 : 1.
- Step a) of the process according to the present invention is often carried out at a temperature in the range of from ambient temperature, such as from about 15°C to about 30°C, up to the reflux temperature of the solvent employed, such as from about 50°C to about 120°C.
- Step b) of the process according to the present invention is often carried out at a temperature in the range of from about -25°C to ambient temperature, such as from about 15°C to about 30°C, such as from about -20°C to about 0°C. Temperatures in the range of from -20°C to -10°C are especially preferred.
- substantially anhydrous reaction conditions are employed.
- the process of the present invention is carried out under an inert atmosphere, such as a nitrogen or argon atmosphere.
- the product compound of formula R 1 -Y 1 -P(NR 2 R 3 ) 2 is advantageously separated from the reaction mixture by distillation, and especially preferably by wiped-film distillation.
- a particularly preferred embodiment of the present invention comprises a process for the preparation of ⁇ [(CH 3 ) 2 CH] 2 N ⁇ 2 -P-O-CH 2 CH 2 CN, which comprises a) reacting PCI 3 with [(CH 3 ) 2 CH] 2 N-H in toluene to form ⁇ [(CH 3 ) 2 CH] 2 N ⁇ 2 -P-CI; and b) reacting ⁇ [(CH 3 ) 2 CH] 2 N ⁇ 2 -P-CI with HO-CH 2 CH 2 CN in toluene to form ⁇ [(CH 3 ) 2 CH] 2 N ⁇ 2 -P- O-CH 2 CH 2 CN.
- the dried amine/toluene mixture was distilled into an oven-dried nitrogen-flushed, 3L, 4-neck round-bottom flask equipped with a mechanical stirrer, a 100 mL, pressure-equalizing addition funnel, a thermowell and a condenser attached to an N 2 bubbler, and allowed to cool to ambient temperature (ca. 17°C).
- Phosphorus trichloride (99.999%, 100g) was charged from the addition funnel over approximately 30 min. The temperature of the mixture was observed to rise ca. 25°C. The mixture was heated to reflux (100-110°C) and stirred for 24 hrs; the mixture becoming quite thick with precipitated diisopropylamine hydrochloride.
- Triethylamine (49.4g), toluene (649.5g) and calcium hydride (5g) were charged to a nitrogen-flushed, 1 L round-bottom flask equipped with a magnetic stirrer and cold-finger distillation head attached to an N 2 bubbler and the mixture heated under total reflux (98°C) for 2 hrs to remove all traces of water.
- the dried mixture was distilled into an oven-dried, nitrogen-flushed, 2L 3-neck, jacketed round-bottom flask equipped with a mechanical stirrer, a thermowell and a 50 ml_ pressure-equalizing addition funnel attached to an N 2 bubbler and the mixture cooled to below -15°C using a circulating chiller pumping ethylene glycol/water through the reactor jacket and the product of step (a) (130.3g) added.
- Neat 2-cyanoethanol (39.9g) was added from the addition funnel over approximately 30 min, keeping the temperature below -10°C.
- the mixture was stirred under nitrogen for 18 hrs at -15 to -18°C to complete the reaction.
- the mixture was warmed to ambient temperature and pressure-filtered under nitrogen to remove triethylamine hydrochloride salt, all equipment being oven-dried before use.
- the toluene was stripped out on a rotary evaporator (bath temperature ca. 50°C).
- the vacuum was broken with nitrogen and the distillation vessel connected via a cold trap directly to a vacuum pump and residual toluene removed to yield 139.5g of crude product.
- the crude product was distilled through a wiped-film evaporator (heated zone 70°C; pressure 0.008 mmHg) over 1.5 hrs to afford 115.9g of product.
- the product can be further purified by flash chromatography, for example using pentane and dry basic alumina, and additional wiped-film distillations.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
Abstract
A process for the preparation of a compound of formula R1-Y1-P(NR2R3 )2 is provided. The process comprises reacting a compound of formula PX3 with a compound of formula HNR2R3 to form a compound of formula X-P(NR2R3)2; and reacting the compound of formula X-P(NR2R3)2 with a compound of formula R1-Y1-H in the presence of a hydrocarbon solvent to form the compound of formula R1-Y1-P(NR2R3 )2. R1 represents a phosphorus protecting group; R2 and R3 each independently represent an alkyl, preferably a C1-6alkyl, group, or R2 and R3 are joined, together with the N to which they are attached, to form a 5-7 membered ring; Y1 represents O or S, preferably O; and X represents a halogen, preferably Cl. The preferred solvent is toluene.
Description
PROCESS FOR THE PREPARATION OF PHOSPHITYLAT1ON AGENTS
The present invention concerns a process for the preparation of phosphitylation agents.
Synthetic oligonucleotides are important diagnostic tools for the detection of genetic and viral diseases. In addition, oligonucleotides and modified oligonucleotides are of interest as therapeutic candidates that inhibit gene expression or protein function. Large scale synthesis of oligonucleotides for use as therapeutic candidates has become increasingly important since FDA approval of an oligonucleotide analog for the treatment of cytomegalovirus (CMV), and several other oligonucleotide analogs are currently in clinical trials. Kilogram quantities of a purified oligonucleotide analog are needed for each clinical trial.
The principal method currently employed for the preparation of oligonucleotide is the phosphoramidite approach. The increasing demand for larger quantities of oligonucleotides has correspondingly increased demand for phosphoramidite compounds. Phosphoramidite compounds are commonly prepared by phosphitylation of a nucleoside with a phosphitylation agent in the presence of an activator. Accordingly, there has been a corresponding increase in demand for phosphitylation agents. Improved or alternative processes for the preparation of phosphitylation agents are therefore needed.
According to the present invention, there is provided a process for the preparation of a compound of formula R1-Y1-P(NR2R3)2 which comprises: a) reacting a compound of formula PX3 with a compound of formula HNR2R3 to form a compound of formula X-P(NR2R3)2; and b) reacting the compound of formula X-P(NR2R3)2 with a compound of formula R1-Y1-H in the presence of a solvent to form the compound of formula R1-Y1-P(NR2R3)2 wherein
R1 represents a phosphorus protecting group;
R2 and R3 each independently represent an alkyl, preferably a C^ alkyl, group, or R2 and
R3 are joined, together with the N to which they are attached, to form a 5-7 membered ring; Y1 represents O or S, preferably O; and
X represents a halogen, preferably Cl; characterised in that the solvent employed in reaction b) is a hydrocarbon solvent.
Phosphorus protecting groups represented by R1 are commonly cleavable phosphorus protecting groups employed in oligonucleotide synthesis, for example substituted or unsubstituted aliphatic groups, such as a methyl group, -CH2CH2-
Si(CH3)2C6H5l -CH2CH2-S(O)2-CH2CH3l -CH2CH2-C6H4-NO2 and preferably a group of formula -CH2CH2CN; or substituted or unsubstituted aromatic groups, such as a phenyl or
substituted phenyl, for example a 4-chlorophenyl, 2-chlorophenyl, 2-nitrophenyl or 4- nitrophenyl group.
Compounds of formula R1-Y1-H are preferably selected based on the nature of the compound it is desired to produce. An especially preferred compound of formula R1-Y1-H is 2-cyanoethanol.
In the compounds prepared by the process of the present invention, it is preferred that R2 and R3 are the same. It is particularly preferred that both R2 and R3 are -CH(CH3)2 groups. It is especially preferred that Y1 is O and R1 is -CH2CH2CN.
Examples of compounds which can be prepared by the process of the present invention include O-β-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, (commonly known as "tetraphos"), O-β-cyanoethyl-N,N,N',N'-tetramethylphosphorodiamidite, O-β- cyanoethyl-N,N,N',N'-tetraethylphosphorodiamidite, bis (N,N-diisopropylamino)-2- methyltrifluoroacetyiamino-ethoxyphosphine, bis (N,N-diisopropylamino)-2- diphenylmethylsilylethoxyphosphine and O-β-cyanoethyl-bis (N-morpholino) phosphorodiamidite.
Hydrocarbon solvents that can be employed in the process of the present invention include aliphatic and aromatic hydrocarbons. Examples of aliphatic hydrocarbons include pentane, hexane and petroleum ethers. Examples of aromatic hydrocarbons include benzene, toluene, xylene and mesitylene. Toluene is the most preferred solvent.
In many preferred embodiments, reaction a) takes place in the presence of the same solvent as reaction b), and most preferably in the presence of toluene.
The reaction between the compound of formula X-P(NR2R3)2 and the compound of formula R1-Y1-H preferably takes place in the presence of a base. Bases which can be employed include inorganic bases, such as sodium carbonate, and organic bases. Organic bases are preferred. Examples of organic bases include aromatic amines such as pyridine, and inorganic amines, such as alkylamines, preferably trialkylamines, such as tr C-MalkyOamines, and most preferably triethylamine.
The mole ratio of compound of formula PX3 to compound of formula HNR2R3 in step a) is commonly selected to be in the range of from about 1 : 1 to about 10 : 1 , and preferably from about 3 : 1 to about 6 : 1.
The mole ratio of compound of formula X-P(NR2R3)2 to compound of formula R1- Y1-H in step b) is commonly selected to be in the range of from about 1 : 1 to about 5 : 1 , with mole ratios in the range of from 1 : 1 to 1.5 : 1 being especially preferred. When a base is employed, the mole ratio of base to compound of formula
X-P(NR2R3)2 is often in the range of from about 0.75 : 1 to 2 : 1 , and preferably from about 1 : 1 to 1.3 : 1.
Step a) of the process according to the present invention is often carried out at a temperature in the range of from ambient temperature, such as from about 15°C to about
30°C, up to the reflux temperature of the solvent employed, such as from about 50°C to about 120°C.
Step b) of the process according to the present invention is often carried out at a temperature in the range of from about -25°C to ambient temperature, such as from about 15°C to about 30°C, such as from about -20°C to about 0°C. Temperatures in the range of from -20°C to -10°C are especially preferred.
Advantageously, substantially anhydrous reaction conditions are employed. In many embodiments the process of the present invention is carried out under an inert atmosphere, such as a nitrogen or argon atmosphere. The product compound of formula R1-Y1-P(NR2R3)2 is advantageously separated from the reaction mixture by distillation, and especially preferably by wiped-film distillation. A particularly preferred embodiment of the present invention comprises a process for the preparation of {[(CH3)2CH]2N}2-P-O-CH2CH2CN, which comprises a) reacting PCI3 with [(CH3)2CH]2N-H in toluene to form {[(CH3)2CH]2N}2-P-CI; and b) reacting {[(CH3)2CH]2N}2-P-CI with HO-CH2CH2CN in toluene to form {[(CH3)2CH]2N}2-P- O-CH2CH2CN.
The present invention is illustrated without limitation by the following Example. Example Step a) Diisopropylamine (383g), toluene (1087g) and calcium hydride (10g) were charged to a nitrogen-flushed, 2 L round-bottom flask equipped with a magnetic stirrer and cold- finger distillation head attached to an N2 bubble and the mixture heated under total reflux (98°C) for 2 hrs to remove all traces of water. The dried amine/toluene mixture was distilled into an oven-dried nitrogen-flushed, 3L, 4-neck round-bottom flask equipped with a mechanical stirrer, a 100 mL, pressure-equalizing addition funnel, a thermowell and a condenser attached to an N2 bubbler, and allowed to cool to ambient temperature (ca. 17°C). Phosphorus trichloride (99.999%, 100g) was charged from the addition funnel over approximately 30 min. The temperature of the mixture was observed to rise ca. 25°C. The mixture was heated to reflux (100-110°C) and stirred for 24 hrs; the mixture becoming quite thick with precipitated diisopropylamine hydrochloride. The mixture was cooled to ambient temperature and pressure-filtered under nitrogen to remove the amine salt, all equipment being oven-dried before use. The filter cake was washed with 217g of toluene, and the combined filtrate and washings distilled in vacuo to remove most of the toluene, the mixture becoming a slush as solids precipitate. When no more toluene distilled, the vacuum was broken with nitrogen and the distillation vessel connected via a cold trap directly to a vacuum pump. Residual toluene was pumped out until the solid product, {[(CH3)2CH]2N}2-P-CI, reached constant weight. This material, 96-97 area-% active by 31P N R was used in Step b) without further purification.
Step b)
Triethylamine (49.4g), toluene (649.5g) and calcium hydride (5g) were charged to a nitrogen-flushed, 1 L round-bottom flask equipped with a magnetic stirrer and cold-finger distillation head attached to an N2 bubbler and the mixture heated under total reflux (98°C) for 2 hrs to remove all traces of water. The dried mixture was distilled into an oven-dried, nitrogen-flushed, 2L 3-neck, jacketed round-bottom flask equipped with a mechanical stirrer, a thermowell and a 50 ml_ pressure-equalizing addition funnel attached to an N2 bubbler and the mixture cooled to below -15°C using a circulating chiller pumping ethylene glycol/water through the reactor jacket and the product of step (a) (130.3g) added. Neat 2-cyanoethanol (39.9g) was added from the addition funnel over approximately 30 min, keeping the temperature below -10°C. The mixture was stirred under nitrogen for 18 hrs at -15 to -18°C to complete the reaction. The mixture was warmed to ambient temperature and pressure-filtered under nitrogen to remove triethylamine hydrochloride salt, all equipment being oven-dried before use. The toluene was stripped out on a rotary evaporator (bath temperature ca. 50°C). The vacuum was broken with nitrogen and the distillation vessel connected via a cold trap directly to a vacuum pump and residual toluene removed to yield 139.5g of crude product. The crude product was distilled through a wiped-film evaporator (heated zone 70°C; pressure 0.008 mmHg) over 1.5 hrs to afford 115.9g of product. If desired, the product can be further purified by flash chromatography, for example using pentane and dry basic alumina, and additional wiped-film distillations.
Claims
1. A process for the preparation of a compound of formula R1-Y1-P(NR2R3)2 which comprises: a) reacting a compound of formula PX3 with a compound of formula HNR2R3 to form a compound of formula X-P(NR2R3)2; and b) reacting the compound of formula X-P(NR2R3)2 with a compound of formula R1-Y1-H in the presence of a solvent to form the compound of formula R1-Y1-P(NR2R3)2; wherein R1 represents a phosphorus protecting group;
R2 and R3 each independently represent an alkyl group, or R2 and R3 are joined, together with the N to which they are attached, to form a 5-7 membered ring;
Y1 represents O or S; and
X represents a halogen; characterised in that the solvent employed in reaction b) is a hydrocarbon solvent.
2. A process according to claim 1 , wherein the reaction between the compound of formula PX3 and the compound of formula HNR2R3 in step a) takes place in the presence of the same solvent employed for the reaction between the compound of formula X- P(NR2R3)2 and the compound of formula R1-Y1-H in step b).
3. A process according to claim 1 or claim 2, wherein R1 represents a methyl group, a group of formula -CH2CH2-Si(CH3)2C6H5) -CH2CH2-S(O)2-CH2CH3 or -CH2CH2-C6H4-NO2, a group of formula -CH2CH2CN, or a phenyl, 4-chlorophenyl, 2-chlorophenyl, 2-nitrophenyl or 4-nitrophenyl group.
4. A process according to claim 3, wherein R1 represents a group of formula -CH2CH2CN and Y1 represents O.
5. A process according to any preceding claim, wherein R2 and R3 each independently represent a Cι.6 alkyl group.
6. A process according to claim 5, wherein R2 and R3 represent isopropyl groups.
7. A process according to any preceding claim, wherein Y1 represents O.
8. A process according to any preceding claim, wherein X represents Cl.
9. A process according to any preceding claim, wherein the hydrocarbon solvent is toluene.
10. A process according to any preceding claim, wherein the reaction between the compound of formula X-P(NR2R3)2 and the compound of formula R1-Y1-H in step b) takes place in the presence of a base.
11. A process according to claim 10, wherein the base is a tπXC^alky amine.
12. A process for the preparation of {[(CHs^CH^N^-P-O-C^C^CN, which comprises a) reacting PCI3 with [(CH3)2CH]2N-H in toluene to form {[(CH3)2CH]2N}2-P-CI; and b) reacting {[(CH3)2CH]2N}2-P-CI with HO-CH2CH2CN in toluene to form {[(CH3)2CH]2N}2-P- O-CH2CH2CN.
13. A process according to any preceding claim, wherein substantially anhydrous reaction conditions are employed.
14. A process for the preparation of a compound of formula R1-Y1-P(NR R3)2 which comprises reacting a compound of formula X-P(NR2R3)2 with a compound of formula R1- Y1-H in the presence of a solvent to form the compound of formula R1-Y1-P(NR2R3)2 wherein
R1 represents a phosphorus protecting group;
R2 and R3 each independently represent an alkyl group, or R2 and R3 are joined, together with the N to which they are attached, to form a 5-7 membered ring; Y1 represents O or S; and
X represents a halogen; characterised in that the solvent is a hydrocarbon solvent.
15. A process according to claim 14, wherein R1 represents NCCH2CH2-; Y1 represents O; R2 and R3 are each isopropyl, X is chloro, and the hydrocarbon solvent is toluene.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT03780393T ATE511513T1 (en) | 2002-12-17 | 2003-12-16 | METHOD FOR PRODUCING PHOSPHITYLATION AGENTS |
| JP2004559906A JP2006509813A (en) | 2002-12-17 | 2003-12-16 | Process for producing a phosphitylating agent |
| KR1020057011086A KR101167297B1 (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
| EP03780393A EP1575965B1 (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
| CN2003801062448A CN1726220B (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
| CA2510473A CA2510473C (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
| AU2003288555A AU2003288555A1 (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
| US10/539,210 US7560555B2 (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US43378802P | 2002-12-17 | 2002-12-17 | |
| US60/433,788 | 2002-12-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2004055030A1 true WO2004055030A1 (en) | 2004-07-01 |
Family
ID=32595244
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB2003/005473 WO2004055030A1 (en) | 2002-12-17 | 2003-12-16 | Process for the preparation of phosphitylation agents |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US7560555B2 (en) |
| EP (1) | EP1575965B1 (en) |
| JP (1) | JP2006509813A (en) |
| KR (1) | KR101167297B1 (en) |
| CN (1) | CN1726220B (en) |
| AT (1) | ATE511513T1 (en) |
| AU (1) | AU2003288555A1 (en) |
| CA (1) | CA2510473C (en) |
| WO (1) | WO2004055030A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62212395A (en) | 1986-03-11 | 1987-09-18 | Nippon Zeon Co Ltd | Production of phosphorus amide compound |
-
2003
- 2003-12-16 CN CN2003801062448A patent/CN1726220B/en not_active Expired - Lifetime
- 2003-12-16 AT AT03780393T patent/ATE511513T1/en not_active IP Right Cessation
- 2003-12-16 EP EP03780393A patent/EP1575965B1/en not_active Expired - Lifetime
- 2003-12-16 US US10/539,210 patent/US7560555B2/en not_active Expired - Lifetime
- 2003-12-16 AU AU2003288555A patent/AU2003288555A1/en not_active Abandoned
- 2003-12-16 CA CA2510473A patent/CA2510473C/en not_active Expired - Lifetime
- 2003-12-16 WO PCT/GB2003/005473 patent/WO2004055030A1/en active Application Filing
- 2003-12-16 JP JP2004559906A patent/JP2006509813A/en active Pending
- 2003-12-16 KR KR1020057011086A patent/KR101167297B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62212395A (en) | 1986-03-11 | 1987-09-18 | Nippon Zeon Co Ltd | Production of phosphorus amide compound |
Non-Patent Citations (16)
| Title |
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| BURGADA R.: "Les réactions du carbonyle avec les aminophosphines X. Effet de substituant dans les réactions d'échange.", BULLETIN DE LA SOCIETE CHIMIQUE DE FRANCE., no. 1, 1971, FRSOCIETE FRANCAISE DE CHIMIE. PARIS., pages 136 - 143, XP002272469 * |
| BURGADA, BULLETIN DE LA SOCIETE CHIMIQUE DE FRANCE, 1971, pages 136 - 143 |
| DATABASE CHEMABS [online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; TAWARA, SHINICHIRO ET AL: "Phosphorodiamidous acid ester derivatives", XP002272471, retrieved from STN Database accession no. 109:149797 * |
| HAMAMOTO ET AL., CHEMISTRY LETTERS, 1986, pages 1401 - 1404 |
| HAMAMOTO S. ET AL.: "New approach to the synthesis of deoxyribonucleoside phosphoramidite derivatives", CHEMISTRY LETTERS., no. 8, 1986, JPCHEMICAL SOCIETY OF JAPAN. TOKYO., pages 1401 - 1404, XP002272465 * |
| HEINICKE ET AL., JOURNAL FUER PRAKTISCHE CHEMIE, vol. 325, no. 3, 1983, pages 511 - 516 |
| HEINICKE J. ET AL.: "Synthese von o-Halogenphenoxy-, o-Halogenphenylthio- und o-Halogen-methylanilinoderivaten der IVb- und Vb-Elemente", JOURNAL FUER PRAKTISCHE CHEMIE., vol. 325, no. 3, 1983, DEWILEY VCH, WEINHEIM., pages 511 - 516, XP008028030 * |
| KIBARDIN A.M. ET AL.: "Synthesis of ethyl bis(N-butyl-N-isobutenylamido)phosphite and reaction with alpha- chloroacetaldehydes", BULLETIN OF THE ACADEMY OF SCIENCES OF THE USSR. DIVISION OF CHEMICAL SCIENCE., vol. 31, no. 3, 20 September 1982 (1982-09-20), USCONSULTANTS BUREAU. NEW YORK., pages 625 - 627, XP002272468 * |
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| KRUCZYNSKI L.J. ET AL.: "Phenylfluorophosphoranes: axial-equatorial fluorine exchange in RC6H4PF3H and intermolecular exchange in the PhPF2(H)OMe-MeOH-base system", CANADIAN JOURNAL OF CHEMISTRY., vol. 68, no. 3, 1990, CANATIONAL RESEARCH COUNCIL. OTTAWA., pages 488 - 491, XP002272466 * |
| MITSUNOBU O. ET AL.: "Preparation of carboxylic esters and phosphoric esters by the activation of alcohols", BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN., vol. 44, no. 12, 1971, JPJAPAN PUBLICATIONS TRADING CO. TOKYO., pages 3427 - 3430, XP002272470 * |
| MITSUNOBU, BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, vol. 44, no. 12, 1971, pages 3437 - 3430 |
| NIFANT'EV E.E. ET AL.: "New aspects of the chemistry of pyrrolides of tervalent phosphorus acids", RUSSIAN JOURNAL OF GENERAL CHEMISTRY., vol. 62, no. 7, 20 December 1992 (1992-12-20), USCONSULTANTS BUREAU., pages 1201 - 1208, XP002272467 * |
| NIFANT'EV ET AL., RUSSIAN JOURNAL OF GENERAL CHEMISTRY, vol. 62, no. 7, 1992, pages 1201 - 1208 |
| WILK A ET AL: "The 4-oxopentyl group as a labile phosphate/thiophosphate protecting group for synthetic oligodeoxyribonucleotides", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 42, no. 33, 13 August 2001 (2001-08-13), pages 5635 - 5639, XP004295829, ISSN: 0040-4039 * |
| WILK ET AL., TETT. LETT., vol. 42, no. 33, 2001, pages 5635 - 5639 |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20050085705A (en) | 2005-08-29 |
| US20060173187A1 (en) | 2006-08-03 |
| CA2510473A1 (en) | 2004-07-01 |
| CN1726220B (en) | 2010-04-28 |
| US7560555B2 (en) | 2009-07-14 |
| AU2003288555A1 (en) | 2004-07-09 |
| JP2006509813A (en) | 2006-03-23 |
| CN1726220A (en) | 2006-01-25 |
| ATE511513T1 (en) | 2011-06-15 |
| CA2510473C (en) | 2012-02-21 |
| EP1575965A1 (en) | 2005-09-21 |
| KR101167297B1 (en) | 2012-07-23 |
| EP1575965B1 (en) | 2011-06-01 |
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