EP3016964A1 - Method for incorporating protecting acetal and acetal ester groups, and its application for the protection of hydroxyl function - Google Patents
Method for incorporating protecting acetal and acetal ester groups, and its application for the protection of hydroxyl functionInfo
- Publication number
- EP3016964A1 EP3016964A1 EP14723139.3A EP14723139A EP3016964A1 EP 3016964 A1 EP3016964 A1 EP 3016964A1 EP 14723139 A EP14723139 A EP 14723139A EP 3016964 A1 EP3016964 A1 EP 3016964A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- group
- reaction
- alkyl
- acetal
- hydroxyl
- 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- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 100
- 125000002887 hydroxy group Chemical group [H]O* 0.000 title claims abstract description 59
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 title claims abstract 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 141
- 229910021627 Tin(IV) chloride Inorganic materials 0.000 claims abstract description 42
- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 claims abstract description 42
- 150000001875 compounds Chemical class 0.000 claims abstract description 41
- 238000010348 incorporation Methods 0.000 claims abstract description 20
- 150000003833 nucleoside derivatives Chemical class 0.000 claims abstract description 17
- 239000000010 aprotic solvent Substances 0.000 claims abstract description 14
- 150000002894 organic compounds Chemical class 0.000 claims abstract description 6
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 42
- -1 nitrile compounds Chemical class 0.000 claims description 40
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 38
- 239000002904 solvent Substances 0.000 claims description 38
- 125000000217 alkyl group Chemical group 0.000 claims description 29
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 23
- 239000000203 mixture Chemical class 0.000 claims description 23
- 229910052736 halogen Inorganic materials 0.000 claims description 16
- 150000002367 halogens Chemical class 0.000 claims description 16
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 15
- 125000001424 substituent group Chemical group 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 125000000266 alpha-aminoacyl group Chemical group 0.000 claims description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 8
- 125000003118 aryl group Chemical group 0.000 claims description 8
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 8
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 8
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 5
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 4
- 125000002877 alkyl aryl group Chemical group 0.000 claims description 4
- 239000003849 aromatic solvent Chemical class 0.000 claims description 4
- 150000004292 cyclic ethers Chemical class 0.000 claims description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 4
- 125000000468 ketone group Chemical group 0.000 claims description 4
- 125000001624 naphthyl group Chemical group 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 4
- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 claims description 4
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- 229940035893 uracil Drugs 0.000 claims description 4
- 150000004250 monothioacetals Chemical class 0.000 claims description 3
- 150000003254 radicals Chemical class 0.000 claims description 3
- YQTCQNIPQMJNTI-UHFFFAOYSA-N 2,2-dimethylpropan-1-one Chemical group CC(C)(C)[C]=O YQTCQNIPQMJNTI-UHFFFAOYSA-N 0.000 claims description 2
- 229930024421 Adenine Natural products 0.000 claims description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 2
- 229960000643 adenine Drugs 0.000 claims description 2
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 2
- 229940104302 cytosine Drugs 0.000 claims description 2
- 125000005441 o-toluyl group Chemical group [H]C1=C([H])C(C(*)=O)=C(C([H])=C1[H])C([H])([H])[H] 0.000 claims description 2
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 claims description 2
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- 125000000037 tert-butyldiphenylsilyl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1[Si]([H])([*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 2
- 229940113082 thymine Drugs 0.000 claims description 2
- 125000001302 tertiary amino group Chemical group 0.000 claims 4
- 229910008433 SnCU Inorganic materials 0.000 claims 1
- 239000002777 nucleoside Substances 0.000 abstract description 30
- 230000015572 biosynthetic process Effects 0.000 abstract description 29
- 238000003786 synthesis reaction Methods 0.000 abstract description 27
- 230000006819 RNA synthesis Effects 0.000 abstract description 7
- 125000003835 nucleoside group Chemical group 0.000 abstract description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 48
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 46
- 239000000047 product Substances 0.000 description 45
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 41
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 40
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 38
- 239000000243 solution Substances 0.000 description 37
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 35
- 238000005481 NMR spectroscopy Methods 0.000 description 30
- 229940045145 uridine Drugs 0.000 description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 24
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 24
- 235000017557 sodium bicarbonate Nutrition 0.000 description 24
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 24
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 23
- 150000001241 acetals Chemical class 0.000 description 23
- 125000006239 protecting group Chemical group 0.000 description 23
- 238000004611 spectroscopical analysis Methods 0.000 description 22
- 239000001569 carbon dioxide Substances 0.000 description 20
- 229910002092 carbon dioxide Inorganic materials 0.000 description 20
- 238000001816 cooling Methods 0.000 description 18
- 230000000903 blocking effect Effects 0.000 description 17
- 239000012047 saturated solution Substances 0.000 description 17
- AOJFQRQNPXYVLM-UHFFFAOYSA-N pyridin-1-ium;chloride Chemical compound [Cl-].C1=CC=[NH+]C=C1 AOJFQRQNPXYVLM-UHFFFAOYSA-N 0.000 description 16
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 14
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 13
- 239000011541 reaction mixture Substances 0.000 description 13
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 12
- 150000001412 amines Chemical group 0.000 description 12
- 239000003480 eluent Substances 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 239000007789 gas Substances 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 239000012044 organic layer Substances 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 8
- 229920006395 saturated elastomer Polymers 0.000 description 8
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 6
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 229910052708 sodium Inorganic materials 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 description 6
- 235000011152 sodium sulphate Nutrition 0.000 description 6
- 229910021653 sulphate ion Inorganic materials 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- VDSCGIJBKRIUDF-UHFFFAOYSA-N (4-chlorophenyl)sulfanylmethyl benzoate Chemical compound C1=CC(Cl)=CC=C1SCOC(=O)C1=CC=CC=C1 VDSCGIJBKRIUDF-UHFFFAOYSA-N 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000002342 ribonucleoside Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- GPENIBIGLJGORG-UHFFFAOYSA-N (4-chlorophenyl)sulfanylmethanol Chemical compound OCSC1=CC=C(Cl)C=C1 GPENIBIGLJGORG-UHFFFAOYSA-N 0.000 description 4
- GFJKNGXJCDLRAM-UHFFFAOYSA-N 3-(chloromethoxy)propanenitrile Chemical compound ClCOCCC#N GFJKNGXJCDLRAM-UHFFFAOYSA-N 0.000 description 4
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 4
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N acetic acid anhydride Natural products CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 4
- 229960005305 adenosine Drugs 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 238000007711 solidification Methods 0.000 description 4
- 230000008023 solidification Effects 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 150000003512 tertiary amines Chemical group 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 125000004430 oxygen atom Chemical group O* 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- LRQORJSCZDMZEM-UHFFFAOYSA-N (4-chlorophenyl)sulfanylmethyl 2,2-dimethylpropanoate Chemical compound CC(C)(C)C(=O)OCSC1=CC=C(Cl)C=C1 LRQORJSCZDMZEM-UHFFFAOYSA-N 0.000 description 2
- UHXSPWSGMZPCLH-UHFFFAOYSA-N (4-methylphenyl)sulfanylmethyl 2,2-dimethylpropanoate Chemical compound CC1=CC=C(SCOC(=O)C(C)(C)C)C=C1 UHXSPWSGMZPCLH-UHFFFAOYSA-N 0.000 description 2
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 2
- JVSFQJZRHXAUGT-UHFFFAOYSA-N 2,2-dimethylpropanoyl chloride Chemical compound CC(C)(C)C(Cl)=O JVSFQJZRHXAUGT-UHFFFAOYSA-N 0.000 description 2
- 108091027075 5S-rRNA precursor Proteins 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 101100206458 Mus musculus Them4 gene Proteins 0.000 description 2
- LQZMLBORDGWNPD-UHFFFAOYSA-N N-iodosuccinimide Chemical compound IN1C(=O)CCC1=O LQZMLBORDGWNPD-UHFFFAOYSA-N 0.000 description 2
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 125000004036 acetal group Chemical group 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- YCACYQHCNYLRKH-UHFFFAOYSA-N benzoyloxymethylsulfanylmethoxymethylsulfanylmethyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCSCOCSCOC(=O)C1=CC=CC=C1 YCACYQHCNYLRKH-UHFFFAOYSA-N 0.000 description 2
- 125000006367 bivalent amino carbonyl group Chemical group [H]N([*:1])C([*:2])=O 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 150000003983 crown ethers Chemical class 0.000 description 2
- RJGHQTVXGKYATR-UHFFFAOYSA-L dibutyl(dichloro)stannane Chemical compound CCCC[Sn](Cl)(Cl)CCCC RJGHQTVXGKYATR-UHFFFAOYSA-L 0.000 description 2
- SPWVRYZQLGQKGK-UHFFFAOYSA-N dichloromethane;hexane Chemical compound ClCCl.CCCCCC SPWVRYZQLGQKGK-UHFFFAOYSA-N 0.000 description 2
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 125000006237 oxymethylenoxy group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- 239000012451 post-reaction mixture Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- GRJJQCWNZGRKAU-UHFFFAOYSA-N pyridin-1-ium;fluoride Chemical compound F.C1=CC=NC=C1 GRJJQCWNZGRKAU-UHFFFAOYSA-N 0.000 description 2
- 239000012429 reaction media Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- YVRNRVQCHUBSGU-UHFFFAOYSA-N (4-methylphenyl)sulfanylmethanol Chemical compound CC1=CC=C(SCO)C=C1 YVRNRVQCHUBSGU-UHFFFAOYSA-N 0.000 description 1
- QRJBTNRZDWGNMD-UHFFFAOYSA-N (4-methylphenyl)sulfanylmethyl benzoate Chemical compound C1=CC(C)=CC=C1SCOC(=O)C1=CC=CC=C1 QRJBTNRZDWGNMD-UHFFFAOYSA-N 0.000 description 1
- OTACXOORCUVHRF-PNHWDRBUSA-N 1-[(2r,3r,4s,5r)-2-ethyl-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione Chemical compound C1=CC(=O)NC(=O)N1[C@]1(CC)O[C@H](CO)[C@@H](O)[C@H]1O OTACXOORCUVHRF-PNHWDRBUSA-N 0.000 description 1
- IBSQPLPBRSHTTG-UHFFFAOYSA-N 1-chloro-2-methylbenzene Chemical compound CC1=CC=CC=C1Cl IBSQPLPBRSHTTG-UHFFFAOYSA-N 0.000 description 1
- FWOXYKSOSYJSAT-UHFFFAOYSA-N 1-chloro-3-[(2-methylphenoxy)methylsulfanyl]benzene Chemical compound CC1=CC=CC=C1OCSC1=CC=CC(Cl)=C1 FWOXYKSOSYJSAT-UHFFFAOYSA-N 0.000 description 1
- VFTFKUDGYRBSAL-UHFFFAOYSA-N 15-crown-5 Chemical compound C1COCCOCCOCCOCCO1 VFTFKUDGYRBSAL-UHFFFAOYSA-N 0.000 description 1
- KJUCPVIVNLPLEE-UHFFFAOYSA-N 2,6-difluoro-n-[2-fluoro-5-[5-[2-[(6-morpholin-4-ylpyridin-3-yl)amino]pyrimidin-4-yl]-2-propan-2-yl-1,3-thiazol-4-yl]phenyl]benzenesulfonamide Chemical compound S1C(C(C)C)=NC(C=2C=C(NS(=O)(=O)C=3C(=CC=CC=3F)F)C(F)=CC=2)=C1C(N=1)=CC=NC=1NC(C=N1)=CC=C1N1CCOCC1 KJUCPVIVNLPLEE-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- BIIYXLIZNQFLAZ-UHFFFAOYSA-N 3-(methylsulfanylmethoxy)propanenitrile Chemical compound CSCOCCC#N BIIYXLIZNQFLAZ-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- DYOFKNUSVMLSTE-UHFFFAOYSA-N [ethylsulfanyl-[ethylsulfanyl-tri(propan-2-yl)silylmethoxy]methyl]-tri(propan-2-yl)silane Chemical compound CCSC([Si](C(C)C)(C(C)C)C(C)C)OC(SCC)[Si](C(C)C)(C(C)C)C(C)C DYOFKNUSVMLSTE-UHFFFAOYSA-N 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical class OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- GGRHYQCXXYLUTL-UHFFFAOYSA-N chloromethyl 2,2-dimethylpropanoate Chemical compound CC(C)(C)C(=O)OCCl GGRHYQCXXYLUTL-UHFFFAOYSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- JGFBRKRYDCGYKD-UHFFFAOYSA-N dibutyl(oxo)tin Chemical compound CCCC[Sn](=O)CCCC JGFBRKRYDCGYKD-UHFFFAOYSA-N 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- AZLPEJUVWWGLHA-UHFFFAOYSA-N ethyl acetate;hexane;methanol Chemical compound OC.CCCCCC.CCOC(C)=O AZLPEJUVWWGLHA-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 238000007327 hydrogenolysis reaction Methods 0.000 description 1
- IKGLACJFEHSFNN-UHFFFAOYSA-N hydron;triethylazanium;trifluoride Chemical compound F.F.F.CCN(CC)CC IKGLACJFEHSFNN-UHFFFAOYSA-N 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910001867 inorganic solvent Inorganic materials 0.000 description 1
- 239000003049 inorganic solvent Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 125000005524 levulinyl group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 125000004092 methylthiomethyl group Chemical group [H]C([H])([H])SC([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- UKLQUFQGBTZEMZ-UHFFFAOYSA-N phenylsulfanylmethanol Chemical class OCSC1=CC=CC=C1 UKLQUFQGBTZEMZ-UHFFFAOYSA-N 0.000 description 1
- PKMUMUIXNICAIL-UHFFFAOYSA-N phenylsulfanylmethyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCSC1=CC=CC=C1 PKMUMUIXNICAIL-UHFFFAOYSA-N 0.000 description 1
- 238000005731 phosphitylation reaction Methods 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical group OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 238000011907 photodimerization Methods 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 229940086066 potassium hydrogencarbonate Drugs 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940058349 sodium levulinate Drugs 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- RDKYCKDVIYTSAJ-UHFFFAOYSA-M sodium;4-oxopentanoate Chemical compound [Na+].CC(=O)CCC([O-])=O RDKYCKDVIYTSAJ-UHFFFAOYSA-M 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 238000000935 solvent evaporation Methods 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- CJTSERYXCRSJAN-UHFFFAOYSA-N tert-butyl-[(4-chlorophenyl)sulfanylmethoxy]-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)OCSC1=CC=C(Cl)C=C1 CJTSERYXCRSJAN-UHFFFAOYSA-N 0.000 description 1
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 description 1
- 150000003555 thioacetals Chemical class 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
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
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
-
- 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
- C07H19/06—Pyrimidine radicals
- C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
-
- 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 is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function.
- the method is applied in particular in the processes of RNA synthesis.
- the method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions.
- protecting groups are used.
- the incorporation of protecting groups must be selective and efficient.
- the linkage between a protecting group and a blocked functional group must be stable in the conditions of successivereactions, and the unblocking must be selective and efficient, e.g. as a result of applying easily accessible and non-toxic reagents, and/or as a result of using physical factors, e.g. heating, irradiation.
- RNA The chemical synthesis of RNA consists of the reaction of condensation of nucleotide units.
- An activated nucleotide unit binds to the free 5 '-OH group of the growing RNA chain.
- an internucleotide linkage is formed between position 3 ' of one nucleotide and position 5 ' of the other nucleotide.
- the remaining reactive centres of the nucleotide are temporarily blocked by protecting groups.
- An appropriate selection of protecting groups enables efficient formation of an internucleotide linkage.
- An appropriate selection of protecting groups, particularly for 2'-hydroxyl functions, and a method for their incorporation and removal, are the main problems associated with the synthesis of RNA chains.
- the unit thus prepared is subjected to a reaction with a phosphitylation reagent in order to incorporate phosphite in position 3 ' .
- Blocking of amine functions of in pyrimidine and purine bases of nucleosides/nucleotides can be performed in optional order depending on a type of amine protection.
- the protecting groupof the 2'-OH function must remain stable until the synthesis of the RNA chain is complete, and its unblocking - which constitutes the final stage of RNA synthesis - cannot adversely affect the synthesized RNA chain.
- a protecting group in position 2' has a direct impact on the reactivity and efficiency of internucleotide linkage formation during the chemical synthesis of RNA fragments, and:
- oligoribonucleotide synthesis An important factor in the processes of oligoribonucleotide synthesis is the stability of protection of hydroxyl and amine functions throughout the entire duration of the process of synthesis - from the stage of preparation of the nucleotide unit used in synthesis, through the successive stages of synthesis, isolation of the final product, and the processes of removal of protecting groups from the final product.
- hydroxyl groups particularly in position 2'
- a range of protecting groups the most common of which are ethers, silyl ethers, acetals and acetal esters.
- Czernecki (3) applied a benzyl group to protect 2'-hydroxyl function.
- the group ensures adequate protection during oligoribonucleotide synthesis, however it is removed by direct hydrogenolysis which may be accompanied by partial hydrogenation of double bonds
- Ohtsuka (4) used a photolabile 2-nitrobenzyl group removable by UV irradiation, which is why the synthesis of the nucleotide unit and the RNA chain must be conducted without the access of light, and furthermore the removal may induce reactions of photodimerization or photodestruction of nucleobases.
- Acetal protecting groups of hydroxyl functions have been known for a long time, however not all of them can be used for protecting the 2'-OH function in the chemical synthesis of RNA.
- the acid-labile tetrahydropyran-l-yl (thp) and methoxytetrahydropyran-4-yl (mthp) groups have known applications as protecting groups of 2'-hydroxyl function.
- the structure of the thp group has a centre of asymmetry at the acetal carbon atom.
- the incorporation of thp group results in the formation of a mixture of diastereoisomers, which might cause necessity of otherwise difficult their separation operation.
- the mthp group has no centre of chirahty. Nevertheless, neither thp nor mthp groups can be employed in the chemical synthesis of RNA if the 5 '-hydroxyl function is blocked with the commonly used DMTr group.
- Reese et al. (8) proposed the application of l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (ctmp) group, stable in the conditions of DMTr group removal.
- Beijer et al. (9) used a l-(2-fluorophenyl)-4-methoxypiperidin-4-yl (fpmp) group which is stable in the conditions of DMTr group removal. All the achiral acetal groups, mthp, ctmp and fpmp, introduce a large steric hindrance, and have a quaternary carbon atom in the a position relative to the 2 '-oxygen atom. Thus, this higher order has an adverse effect on the efficiency of formation of the internucleotide linkage (2).
- a method for protecting the hydroxyl function with acetal or acetal ester derivatives of formaldehyde has been known.
- This protecting groups are sufficiently stable in the acidic environment, as opposed to other acetals.
- These formaldehyde derived acetals exhibit sufficient stability in acidic conditions which are used for DMTr group removal.
- the application of acetalderivatives of formaldehyde makes it possible to use various unblocking conditions depending on the characteristics of the formaldehyde derivative, e.g. their structure allows their removal in conditions other than acidic.
- Acetal or acetal ester derivatives of formaldehyde do not introduce large steric hindrances since they contain a secondary carbon atom in the a-position relative to the 2' oxygen atom. On account of their mixed nature, the groups maintain stability during the chemical synthesis of RNA.
- the patent US5986084 discloses the application of a triisopropylsilyloxymethyl (TOM) group in the synthesis of long RNA fragments.
- the method of incorporating the protecting group in the 2' position in the nucleotide comprises two stages. At the first stage, a substrate with free 3 '- and 2'-hydroxyl groups is subjected to a reaction with dibutyltin(IV) chloride, producing reactive cyclic 5 '-0-(4,4'-dimethoxytrityl)-2',3'-0-dibutylstannateribonucleoside which, at the second stage, is subjected to a reaction with triisopropylsilyloxmethyl chloride.
- the resulting product is a m i xture o f tw o n u c l e o s i d e i s o m e rs , n am e l y 5 '-0-(4,4'-dimethoxytrityl)-2'-0- (triisopropylsilyloxymethyl)nucleoside (5'-0-DMTr-2'-0-TOM-nucleoside) a n d 5 '-0-(4,4'- dimethoxytrityl)-3'-0-(triisopropyl silyloxymethyl)nucleo side (5 -0-DMTr-3'-0-TOM- nucleoside).
- Ohgi et al. 10-10 used a cyanoethoxymethyl (CEM) group for blocking the 2'-OH function in a ribonucleoside.
- CEM cyanoethoxymethyl
- Oghi 9
- the reaction gives rise to reactive cyclic 5'-0-(4,4'-dimethoxytrityl)-2',3'- O-dibutylstannate-ribonucleoside which, at the next stage, is subjected to a reaction with (2- cyanoethoxy)methyl chloride.
- Yoshinobu (11) disclosed a method for incorporation of a 2-cyanoethoxymethyl group protecting the 2'-hydroxyl function of nucleoside, based on the reaction of 3 '- and 5'-protected nucleoside with 2-cyanoethyl methylthiomethyl ether at a very low temperature, in the presence of N-iodosuccinimide and trifluoromethanesulphonic acid.
- the reactions are performed at a temperature of -45 C since higher temperatures may induce the alkylation of pyrimidine bases.
- the method requires the use of expensive reagents.
- the US patent US8536318 discloses the use of a pivaloyloxymethyl group (PivOM) for the protection of hydroxyl function.
- the incorporation of pivaloyloxymethyl group into the hydroxyl function is a two-stage process. It consists of the reaction between a nucleoside protected in position 5'-0-(4,4'-dimethoxytrityl) group and free 3'- and 2'-hydroxyl groups with dibutyltin(IV) oxide, resulting in the formation of cyclic 2',3 '-dibutylstannate-ribonucleoside which, at the next stage, reacts with pivaloyloxymethyl chloride.
- the reaction produces two isomers: 5'-0-(4,4'- dimethoxytrityl)-2'-0-pivaloyloxymethylnucleoside (5'-0-DMTr-2'-0-PivOM-nucleoside) and 5'- 0-(4,4'-dimethoxytrityl)-2'-0-p ivaloylo xym ethyl nuc l eo s i de ( 5 '-0-DMTr-3'-0-PivOM- nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'-0-PivOM- nucleosideis suitable for RNA synthesis.
- the efficiency of obtaining isomer 5 -0-DMTr-2 -0- PivOM-nucleoside is 34-49%.
- the group is compatible with other protecting groups used in the chemical synthesis of oligoribonucleotides, however the disclosed method for its incorporation into the nucleoside is inefficient and involves multiple stages.
- Lackey (12) applied a levulinyloxymethyl (ALE) group for protecting the 2'-hydroxyl function during RNA synthesis.
- ALE levulinyloxymethyl
- the starting component is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)nucleoside which, in a reaction with dimethyl sulphoxide, acetic acid and acetic anhydride, is converted into 5',3'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (methylthiomethyl)nucleoside which, following isolation and purification, is subjected to a reaction with sulphuryl chloride, forming another intermediate product i.e.
- the method comprises several stages and is time consuming, with an overall duration exceeding 30 hours. Furthermore, column chromatography and expensive crown ethers markedly increase the costs of synthesis.
- the purpose of the invention was development of a simple and effective method for incorporation of acetal and acetal ester groups protecting the hydroxyl function in order to use in particular thus blocked compounds in further chemical reactions and to protect the hydroxyl function of the 2' group in nucleosides.
- the present invention is a method for incorporation of an acetal or acetal ester group to protect hydroxyl function.
- the method according to the invention consists of the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, Ri-S-CH 2 -0-R 2 (1) where
- Ri represents a Ci_ 6 alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl; in particular Ri represents -CH 3 -Ph, -Ph(4-Cl), - Ph(4-CH 3 ), -CH 2 Ph
- alkyl-aryl in which the alkyl chain contains Ci_ 5 , whereas aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
- R 3 represents:
- R4, R 5 and R5 are different or the same, and represent a Ci_ 2 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, where the total number of carbon atoms in the group of formula 3 is no less than 6 and no more than 30,
- the method according to the invention can be applied for compounds containing at least one hydroxyl group, and the compounds must be soluble in aprotic solvents.
- the method is only suitable for modifying organic compounds that are soluble in aprotic solvents.
- the method can also be applied for compounds that are insoluble in aprotic solvents, on condition that they are first converted into a form which is soluble in these solvents.
- ribose is insoluble in aprotic solvents, however incorporation of a protecting group, e.g. a silyltert-butyldimethylsilylgroup, into one or two hydroxyl groups will permit ribose dissolution in aprotic solvents.
- the solvents that can be used include halogen derivatives of alkanes, particularly carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents, particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
- the reaction is conducted in anhydrous environments, whereby it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl 4 must be ensured because a part of it is bound by water.
- the organic compound containing at least one hydroxyl group and an appropriate compound of the general formula 1 is dissolved in a solvent, and then SnCl 4 is added to the reaction mixture. It is advantageous to introduce SnCl 4 in the form of a solution in the same solvent as that is the reaction medium or in 1,2-dichloroethane.
- SnCl 4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be exchanged. It is advantageous to use SnCl in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be exchanged most advantageously in an amount from 2.5 to 4.5 moles.
- the ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound of formula 1 to be used in excess ranging from 1 to 8.
- An excess of the compound of formula 1 makes it possible to achieve the highest possible process efficiency within the shortest time.
- the reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture.
- the reaction is conducted in low temperatures, however not lower than the solidification point of the solvent used.
- the higher the temperature the lower the efficiency of obtaining the target product and the greater the quantity of by-products. It is beneficial to conduct the reaction in temperatures from the range from -80 C to 0 C, and most advantageously - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
- the reaction of hydroxyl group blocking involves preparation of a solution of a compound containing a hydroxyl group, and the compound of formula 1, followed by cooling of the mixture to a low temperature. After the cooling procedure, SnCl 4 is introduced, whereupon the reaction is advantageously conducted at a low temperature until the completion of the process.
- it is advantageous to monitor the course of the blocking reaction by thin-layer chromatography on silica gel plates, or by high-performance liquid chromatography (HPLC).
- the duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
- the product is isolated and purified using known methods. Prior to product isolation it is advantageous to neutralize SnCl 4 with a neutralizing agent in an amount of at least 4 molar equivalents of SnCl 4 used. Depending on product stability, neutralization can be performed with:
- a product of hydroxyl function protectionwith an acetal or acetal ester protecting group is an original compound in which the site of the hydroxyl group is occupied by the group of formula 4
- R 2 has the meaning defined above.
- the present invention relates to a method for protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives, consisting of the incorporation of an acetal or acetal ester group.
- the method according to the invention consists of the reaction between the compound of the general formula 5
- B represents radicals of nucleobases, particularly uracil, or appropriately protected residuesof adenine, guanine, cytosine, uracil and thymine,
- Yi and Y 2 are the same or different, and represent groups protecting hydroxyl functions in positions 3' and 5 '; in particular, they are silyl groups: triethylsilyl, tert-butyl-dimethyl- silyl, isopropyl-dimethyl-silyl, tert-butyl-diphenyl-silyl, methyl-diisopropyl-silyl, triphenylsilyl, triisopropylsilyl, methyl-di-tert-butylsilyl
- the blocking compound the compound of the general formula 1 (hereinafter referred to as the blocking compound),
- - i represents a C w alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl; in particular Ri represents -CH 3> -Ph,
- alkyl-aryl in which the alkyl chain contains Ci_ 5 , and aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
- R 3 represents
- ketone group o unsubstituted or substituted phenyl, with substituents including a Ci_ 7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group,
- R4, R 5 and R ⁇ are different or the same, and represent a Ci_ 2 8 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, with the total number of carbon atoms in that group is no less than 6 and no more than 30,
- the method for the protection of the hydroxyl function in nucleosides consists in particular of incorporation of groups of formulas 12 and 13 in position 2'
- the solvents that can be used include halogen derivatives of alkanes, in particular carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents: particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
- the reaction is conducted in anhydrous media but it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl 4 must be ensured because a part of it is bound by water.
- the compound of the general formula 5 or 6, in which substituents have the meaning defined above, and an appropriate blocking compound are dissolved in a solvent, and then SnCl 4 is added to the reaction mixture. It is advantageous to introduce SnCl 4 in the form of a solution in the same solvent as that is the reaction medium or in 1 ,2-dichloroethane. SnCl 4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be protected. It is advantageous to use SnC in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be protected, most advantageously in an amount from 2.5 to 4.5 moles.
- the ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound to be used in excess ranging from 1 to 8. An excess of the compound of the formula 1 makes it possible to achieve the highest possible process efficiency withm the shortest time.
- the reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture, however the temperature of the reaction must not be higher than the temperature of de-protecting the hydroxyl function in positions 3 ' and 5 '. It is advantageous to conduct the reaction in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of the process and the greater the quantity of by-products. It is advantageous to conduct the reaction in the temperature range from -80 C to 0 C, and most advantageous - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
- the reaction of hydroxyl group protection involves preparation of a solution of a compound containing a hydroxyl group which is to be protected, and a blocking compound, followed by the cooling of the mixture to a low temperature. After the cooling procedure, SnCl 4 is introduced, and then the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the reaction by thin-layer chromatography on silica gel plates, or by high- performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
- the product is isolated and purified using known methods, following the same procedure as in the method according to the first aspect of the invention.
- the product of the reaction is the compound of the general formula 14,
- the protecting groups used in the method according to the invention are unblocked in different conditions, on a case-by-case basis, depending on the structure of the group.
- a characteristic property of acetal and acetal ester groups protecting the hydroxyl function is high flexibility in unblocking methods.
- all known chemical unblocking methods can be employed, however due to the specific properties of acetal and acetal ester groups stemming from the nature of these compounds, it is possible to select appropriate conditions which are advantageous for a particular application of the compounds protected with these groups.
- the selection of the most advantageous method of unblocking the hydroxyl function protected with the method according to the invention depends not only on the chemical nature of the blocking group but also on the specific properties of the blockage used.
- the removal of acetal and acetal ester groups can be performed with solutions of inorganic bases, e.g. NaOH, KOH, and organic bases, e.g. amines in organic or inorganic solvents, also in their mixtures. It is advantageous to use weak bases (e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol), which is why during the unblocking there is practically no hydrolysis of mtemucleotide linkages.
- weak bases e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol
- Acetal and acetal ester protecting groups can also be unblocked in acidic conditions, and they are stable in conditions required for the removal of the acid-labile dimethoxytrityl group (DMTr) from the 5 '-hydroxyl position.
- the groups are stable, which is their advantage, towards weak acid solutions used for the unblocking of the 5 '-hydroxyl group blocked with the acid-labile dimethoxytrityl group (DMTr).
- DMTr acid-labile dimethoxytrityl group
- the acetal or acetal ester group protecting the 2'-hydroxyl function is stable, which is a significant factor for the chemical synthesis of the RNA chain.
- Acetal and acetal ester protecting groups can also be removed in reactions that are specific to a particular blocking group, e.g. in a reaction between the carbonyl group of the ieto-ketoester radical (e.g. levulinyl, Lv, H 3 CC(0)CH 2 CH 2 C(0)-,) and the unblocking reagent i.e. hydrazine solution.
- the carbonyl group of the ieto-ketoester radical e.g. levulinyl, Lv, H 3 CC(0)CH 2 CH 2 C(0)-,
- the unblocking reagent i.e. hydrazine solution.
- the subjects of the present invention are new monothioacetals of the general formula 1,
- ⁇ R 2 represents o-toluyl, benzoyl, pivaloyl.
- R 2 has the meanings defined above
- the reaction is conducted in the following manner: 1 eqval of an appropriate compound of formula 17 is dissolved in diethyl ether and combined with 1 egual of an amine. The solution is cooled down to the temperature of 0 C and, on stirring, 1 egualof an appropriate compound of formula 16 is added. On completion of the reaction, the cooling is stopped and a saturated sodium hydrogen carbonate solution is added successively until carbon dioxide no longer evolves from the reaction mixture. The mixture is separated and the organic layer containing the reaction product is dried, following which the solvent is evaporated and the final product is crystallized.
- the method according to the invention is universal and can be applied for the protection of hydroxyl functions with acetal and acetal ester groups.
- the method can be applied for the protection of hydroxyl groups not only in nucleosides and their analogues, but also in alcohols and complex chemical compounds containing a hydroxyl group.
- the method makes use of both known and new compounds, developed specifically for the invention, containing a thioacetal or thioacetal ester group.
- the method according to the invention has a particularly advantageous application in the chemical synthesis of RNA and its analogues.
- the blockage of the hydroxyl function using the method according to the invention is compatible with other protecting groups applied during RNA chain synthesis; for example, it is stable in the conditions of unblocking of the 3'- and 5' -hydroxyl positions with fluoride ions.
- N 6 -phenoxacetyl-2'- benzoyloxymethyl-3',5'-(tetraisopropyldisiloxane-l,3-diyl)adenosine was obtained in the yield of 72%.
- N 2 - tert-butylphenoxacetyl-3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- benzoyloxymethylguanosine was obtained in the yield of 74%.
- a volume of 30 ⁇ (0.3 mmol) of dry benzyl alcohol was transferred into a round-bottom flask and then dissolved in 1 ml of dry 1,2-dichloroethane, then 0.166 g (0.6 mmol) of benzoyloxymethylthiobenzene was added in the presence of 4 A molecular sieves.
- the flask was closed with a septum provided with an argon-filled balloon.
- the mixture was cooled down to the temperature of -25°C and thereafter, on stirring, 0.6 ml of 0.8 M solution of tin(IV) chloride (0.48 mmol) in 1,2-dichloroethane was added.
- the reaction was conducted in argon atmosphere.
- the mixture was stirred magnetically at a temperature of -25°C for 5 hours. Thereafter, the reaction was completed by adding an aqueous solution of sodium hydrogen carbonate until carbon dioxide stopped evolving,then the cooling bath was removed. The white precipitatewas filtered off, and the filtrate was extracted three times with 1,2-dichloroethane (3x3 ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated.
- the raw product was purified on a preparative PLC plate covered with silica gel 60 RP-18, F 25 4, 1 mm, from Merck, using hexane-dichloromethane 2:3 as the mobile phase. The product was extracted with dichloromethane (15 ml). In this manner, benzyloxymethylbenzoyl was obtained in the yield of 56%.
- the product was extracted six times with 5 ml of methylene di chloride. The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product of the reaction was purified in a chromatographic column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (95 :5) as eluents. In this manner, 2'-0-(pivaloyloxymethyl)uridine was obtained in the yield of 84%.
- Example 20 A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution in methanol (2.5 ml) was added. On completion of the unblocking reaction (34 hours), the solvent and amine residue were evaporated from the reaction mixture. The post-reaction mixture was introduced into a chromatography column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (60:40) as eluents. Uridine was isolated as the product of unblocking. NRM analysis confirmed that the compound resulting from the removal of the protecting group is uridine.
- Example 20 A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution
- the raw product was introduced into a chromatography column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (98 :2) as eluents.
- the isolated product of unblocking of the protecting group in position 2 ' is 3 ' ,5 '-0-(tetraisopropyldisiloxane-l,3- diyl)uridine.
- Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
- the isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine.
- Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
- the raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 ⁇ ) from Merck, using methylene dichloride-methanol (60:40) as eluents.
- the collected fraction was evaporated.
- the isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.
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Abstract
The present invention is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function. The method is applied in particular in the processes of RNA synthesis. The method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions. The method according to the invention consists of the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, R1 -S-CH2 -O-R2(1) in the presence of SnCl4, in an aprotic solvent. In the second aspect, the present invention is the method of protecting the hydroxyl function, particularly in position 2', in nucleoside derivatives, based on the incorporation of an acetal or acetal ester group.
Description
Method for incorporating protecting acetal and acetal ester groups, and its application for the protection of hydroxyl function
The present invention is the method for incorporation of acetal and acetal ester groups for protection of hydroxyl function. The method is applied in particular in the processes of RNA synthesis. The method can be employed in the synthesis of nucleosides with acetal and acetal ester groups for the protection of hydroxyl functions.
There are a number of reactions in organic chemistry which involve compounds containing hydroxyl or amine groups, in which it is desirable to block temporarily the reactivity of these groups and, for this reason, techniques for temporary blocking of these functions are employed.
To this aim, protecting groups are used. The incorporation of protecting groups must be selective and efficient. The linkage between a protecting group and a blocked functional group must be stable in the conditions of successivereactions, and the unblocking must be selective and efficient, e.g. as a result of applying easily accessible and non-toxic reagents, and/or as a result of using physical factors, e.g. heating, irradiation.
The chemical synthesis of RNA consists of the reaction of condensation of nucleotide units. An activated nucleotide unit binds to the free 5 '-OH group of the growing RNA chain. In this manner, an internucleotide linkage is formed between position 3 ' of one nucleotide and position 5 ' of the other nucleotide. The remaining reactive centres of the nucleotide, not taking part in a given reaction, are temporarily blocked by protecting groups. An appropriate selection of protecting groups enables efficient formation of an internucleotide linkage.
An appropriate selection of protecting groups, particularly for 2'-hydroxyl functions, and a method for their incorporation and removal, are the main problems associated with the synthesis of RNA chains.
As a rule , the procedure of functional group protection in the synthe sis of oligoribonucleotides proceeds according to the following scheme:
• blocking of the amine functions in pyrimidine and purine bases of the nucleoside,
• blocking of hydroxyl functions, whereby the blocking is generally conducted in the following sequence of steps:
o regioselective blocking of -OH groups in positions 3' and 5',
o blocking of the -OH group in position 2',
o unblocking of -OH groups in positions 3' and 5',
o re-blocking of the -OH group in position 5' - this stage of blocking is performed with 4,4'-dimethoxytntyl (DMTr).
The unit thus prepared is subjected to a reaction with a phosphitylation reagent in order to incorporate phosphite in position 3 ' . Blocking of amine functions of in pyrimidine and purine bases of nucleosides/nucleotides can be performed in optional order depending on a type of amine protection.
The protecting groupof the 2'-OH function must remain stable until the synthesis of the RNA chain is complete, and its unblocking - which constitutes the final stage of RNA synthesis - cannot adversely affect the synthesized RNA chain.
There are a number of ways known toprotecthydroxyl groups in positions 3' and 5', however theseprotecting groupsare useful at intermediate stages, not at the stage of synthesis of RNA chain fragments. A protectionof the hydroxyl function in position 2' is especially important for the process of RNA chain synthesis.
A protecting group in position 2' has a direct impact on the reactivity and efficiency of internucleotide linkage formation during the chemical synthesis of RNA fragments, and:
1. should represent the smallest possible steric hindrance during internucleotide linkage formation. The larger the protecting group in position 2', the lower the efficiency of
internucleotide linkage formation. An important role is played by the order of the carbon atom bound to the oxygen atom in position 2' (1), since it has been proved that the efficiency of in internucleotide linkage formation decreases along with the increase in the order of the carbon atom in position a relative to the oxygen atom in position 2' (2).
2. must be stable in the conditions of RNA chain synthesis: premature unblocking leads to a partial or complete degradation of the RNA chain, and creates the risk of isomerization of internucleotide linkages 3'-5'→ 2'-5'.
3. its incorporation must be selective and efficient.
4. The removal of the blocking group from 2 ' should take place in approximately mildconditions, since basic conditions may induce hydrolysis of the internucleotide linkage, and acidic conditions - isomerization and hydrolysis of the internucleotide linkages.
An important factor in the processes of oligoribonucleotide synthesis is the stability of protection of hydroxyl and amine functions throughout the entire duration of the process of synthesis - from the stage of preparation of the nucleotide unit used in synthesis, through the successive stages of synthesis, isolation of the final product, and the processes of removal of protecting groups from the final product.
The protection of hydroxyl groups, particularly in position 2', is achieved with a range of protecting groups, the most common of which are ethers, silyl ethers, acetals and acetal esters.
1. Ether groups
Czernecki (3) applied a benzyl group to protect 2'-hydroxyl function. The group ensures adequate protection during oligoribonucleotide synthesis, however it is removed by direct hydrogenolysis which may be accompanied by partial hydrogenation of double bonds C5=C6in pyrimidine nucleotide bases.
Ohtsuka (4) used a photolabile 2-nitrobenzyl group removable by UV irradiation, which is why the synthesis of the nucleotide unit and the RNA chain must be conducted without the access of light, and furthermore the removal may induce reactions of photodimerization or photodestruction of nucleobases.
2. Protecting groups based on silyl ethers
Ogilvie (5) applied a f-butyldimethylsilyl group for protection of 2'-hydroxyl function. The group is selectively removed with fluoride ions. The i-butyldimethylsilyl protecting group, however, presents a considerably large steric hindrance which has an adverse effect on the formation of the internucleotide linkage, reducing the efficiency of condensation of nucleotide units.
3. Acetal groups
Acetal protecting groups of hydroxyl functions have been known for a long time, however not all of them can be used for protecting the 2'-OH function in the chemical synthesis of RNA.
The acid-labile tetrahydropyran-l-yl (thp) and methoxytetrahydropyran-4-yl (mthp) groups have known applications as protecting groups of 2'-hydroxyl function.
The structure of the thp group has a centre of asymmetry at the acetal carbon atom. In natural ribonucleosides, which are pure enantiomers, the incorporation of thp group results in the formation of a mixture of diastereoisomers, which might cause necessity of otherwise difficult their separation operation. The mthp group has no centre of chirahty. Nevertheless, neither thp nor mthp groups can be employed in the chemical synthesis of RNA if the 5 '-hydroxyl function is blocked with the commonly used DMTr group.
Reese et al. (8) proposed the application of l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (ctmp) group, stable in the conditions of DMTr group removal.
Beijer et al. (9) used a l-(2-fluorophenyl)-4-methoxypiperidin-4-yl (fpmp) group which is stable in the conditions of DMTr group removal. All the achiral acetal groups, mthp, ctmp and fpmp, introduce a large steric hindrance, and have a quaternary carbon atom in the a position relative to the 2 '-oxygen atom. Thus, this higher order has an adverse effect on the efficiency of formation of the internucleotide linkage (2).
4. Acetal or acetal ester derivatives of formaldehyde
A method for protecting the hydroxyl function with acetal or acetal ester derivatives of formaldehyde has been known. This protecting groups are sufficiently stable in the acidic environment, as opposed to other acetals. These formaldehyde derived acetals exhibit sufficient stability in acidic conditions which are used for DMTr group removal. The application of
acetalderivatives of formaldehyde makes it possible to use various unblocking conditions depending on the characteristics of the formaldehyde derivative, e.g. their structure allows their removal in conditions other than acidic.
Acetal or acetal ester derivatives of formaldehyde do not introduce large steric hindrances since they contain a secondary carbon atom in the a-position relative to the 2' oxygen atom. On account of their mixed nature, the groups maintain stability during the chemical synthesis of RNA.
The patent US5986084 discloses the application of a triisopropylsilyloxymethyl (TOM) group in the synthesis of long RNA fragments. The method of incorporating the protecting group in the 2' position in the nucleotide comprises two stages. At the first stage, a substrate with free 3 '- and 2'-hydroxyl groups is subjected to a reaction with dibutyltin(IV) chloride, producing reactive cyclic 5 '-0-(4,4'-dimethoxytrityl)-2',3'-0-dibutylstannateribonucleoside which, at the second stage, is subjected to a reaction with triisopropylsilyloxmethyl chloride. The resulting product is a m i xture o f tw o n u c l e o s i d e i s o m e rs , n am e l y 5 '-0-(4,4'-dimethoxytrityl)-2'-0- (triisopropylsilyloxymethyl)nucleoside (5'-0-DMTr-2'-0-TOM-nucleoside) a n d 5 '-0-(4,4'- dimethoxytrityl)-3'-0-(triisopropyl silyloxymethyl)nucleo side (5 -0-DMTr-3'-0-TOM- nucleoside). Both isomers require chromatographic separation, as only 5 '-0-DMTr-2'-0-TOM- nucleoside is suitable for RNA synthesis. The yield of synthesis of isomer 5'-0-DMTr-2'-0-TOM- nucleoside is 40%-60%. The unblocking procedure requires the use of fluoride ionsforming appropriate salts and their removal is problematic and calls for an additional oligomer purification procedure.
Ohgi et al. (10) used a cyanoethoxymethyl (CEM) group for blocking the 2'-OH function in a ribonucleoside. The application of this group for 2'-hydroxyl protection turned out to be very efficient, as it allowed obtaining a 1 10-mer RNA chain for the first time
There are two known methods for incorporation of the CEM group. Oghi (9) described a method involving the reaction of blocked 5 '-0-(4,4'-dimethoxytrityl)nucleoside with dibutyltin(IV) chloride. The reaction gives rise to reactive cyclic 5'-0-(4,4'-dimethoxytrityl)-2',3'- O-dibutylstannate-ribonucleoside which, at the next stage, is subjected to a reaction with (2-
cyanoethoxy)methyl chloride. The reaction between 2-cyanoethoxymethyl chloride and 5'-0-(4,4'- dimethoxytrityl)-2',3'-0-dibutyl-stannate-ribonucleoside leads to the formation of two isomers: 5'- 0-(4,4'-dimethoxytrityl)-2'-0-(2-cyanoethoxymethyl)nucle o side (5 '-0-DMTr-2'-0-CEM- nucleoside) and 5 '-0-(4,4'-dimethoxytrityl)-3'-0-(2-cyanoethoxymethyl)nucleoside (5 '-ODMTr- 3'-0-CEM-nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'- O-CEM-nucleoside is suitable for RNA synthesis. The efficiency of obtaining isomer 5' -O-DMTr- 2'-0-CEM-nucleosideis 29-51%. Furthermore, (2-cyanoethoxy)methyl chloride used in the process is a cancerogenic compound.
Yoshinobu (11) disclosed a method for incorporation of a 2-cyanoethoxymethyl group protecting the 2'-hydroxyl function of nucleoside, based on the reaction of 3 '- and 5'-protected nucleoside with 2-cyanoethyl methylthiomethyl ether at a very low temperature, in the presence of N-iodosuccinimide and trifluoromethanesulphonic acid. The reactions are performed at a temperature of -45 C since higher temperatures may induce the alkylation of pyrimidine bases. The method requires the use of expensive reagents.
The US patent US8536318 discloses the use of a pivaloyloxymethyl group (PivOM) for the protection of hydroxyl function. The incorporation of pivaloyloxymethyl group into the hydroxyl function is a two-stage process. It consists of the reaction between a nucleoside protected in position 5'-0-(4,4'-dimethoxytrityl) group and free 3'- and 2'-hydroxyl groups with dibutyltin(IV) oxide, resulting in the formation of cyclic 2',3 '-dibutylstannate-ribonucleoside which, at the next stage, reacts with pivaloyloxymethyl chloride. The reaction produces two isomers: 5'-0-(4,4'- dimethoxytrityl)-2'-0-pivaloyloxymethylnucleoside (5'-0-DMTr-2'-0-PivOM-nucleoside) and 5'- 0-(4,4'-dimethoxytrityl)-2'-0-p ivaloylo xym ethyl nuc l eo s i de ( 5 '-0-DMTr-3'-0-PivOM- nucleoside). Both isomers require chromatographic separation, as only 5'-0-DMTr-2'-0-PivOM- nucleosideis suitable for RNA synthesis. The efficiency of obtaining isomer 5 -0-DMTr-2 -0- PivOM-nucleoside is 34-49%. The group is compatible with other protecting groups used in the chemical synthesis of oligoribonucleotides, however the disclosed method for its incorporation into the nucleoside is inefficient and involves multiple stages.
Lackey (12) applied a levulinyloxymethyl (ALE) group for protecting the 2'-hydroxyl function during RNA synthesis. In the proposed method, the starting component is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)nucleoside which, in a reaction with dimethyl sulphoxide, acetic acid and acetic anhydride, is converted into 5',3'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (methylthiomethyl)nucleoside which, following isolation and purification, is subjected to a reaction with sulphuryl chloride, forming another intermediate product i.e. 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)-2'-0-(chloromethyl)nucleoside which, after solvent evaporation, is dissolved in dichloromethane and combined with the crown ether 15-crown-5 (15- C-5) and sodium levulinate (NaLv). The product of the reaction is 5',3'-0- (tetraisopropyldisiloxane-l,3-diyl)-2'-0-levulinyloxymethyl nucleoside.
The method comprises several stages and is time consuming, with an overall duration exceeding 30 hours. Furthermore, column chromatography and expensive crown ethers markedly increase the costs of synthesis.
Known methods for incorporation of acetal or acetal ester groups to protect the 2'-hydroxyl function of nucleosides are inefficient, expensive, and involve multiple stages, which is why they markedly extend the duration and increase the costs of synthesis, and in many cases require the application of harmful substrates such as 2-cyanoethoxymethyl chloride which is a cancerogenic compound.
The purpose of the invention was development of a simple and effective method for incorporation of acetal and acetal ester groups protecting the hydroxyl function in order to use in particular thus blocked compounds in further chemical reactions and to protect the hydroxyl function of the 2' group in nucleosides.
It was unexpectedly proved that it was possible to conduct a reaction between a hydroxyl group and compounds such as a thioacetal derivative of alcohol or a thioacetal ester derivative of acid in the presence of tin(IV) chloride (SnCl4).
The present invention is a method for incorporation of an acetal or acetal ester group to protect hydroxyl function. The method according to the invention consists of the reaction of an
organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1, Ri-S-CH2-0-R2 (1) where
- Ri represents a Ci_6alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_7alkyl, halogen, aminoacyl; in particular Ri represents -CH3 -Ph, -Ph(4-Cl), - Ph(4-CH3), -CH2Ph
- R2 represents
• a Ci_i5 alkyl;
• alkyl-aryl, in which the alkyl chain contains Ci_5, whereas aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
· a group of the general formula 2
where R3 represents:
o a Ci-15 alkyl;
o a ketone group;
o unsubstituted or substituted phenyl, with substituents including a Ci_7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group.
• a group of the general formula 3
R4
Si R5
Re
(3)
where R4, R5 and R5 are different or the same, and represent a Ci_28 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, where the total number of carbon atoms in the group of formula 3 is no less than 6 and no more than 30,
the presence of SnCl4, in an aprotic solvent.
The method according to the invention can be applied for compounds containing at least one hydroxyl group, and the compounds must be soluble in aprotic solvents.
Due to the properties of SnCl4, the method is only suitable for modifying organic compounds that are soluble in aprotic solvents. The method can also be applied for compounds that are insoluble in aprotic solvents, on condition that they are first converted into a form which is soluble in these solvents. For example, ribose is insoluble in aprotic solvents, however incorporation of a protecting group, e.g. a silyltert-butyldimethylsilylgroup, into one or two hydroxyl groups will permit ribose dissolution in aprotic solvents.
The solvents that can be used include halogen derivatives of alkanes, particularly carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents, particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
The reaction is conducted in anhydrous environments, whereby it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl4 must be ensured because a part of it is bound by water.
In the method according to the invention, the organic compound containing at least one hydroxyl group and an appropriate compound of the general formula 1 is dissolved in a solvent, and then SnCl4 is added to the reaction mixture. It is advantageous to introduce SnCl4 in the form of a solution in the same solvent as that is the reaction medium or in 1,2-dichloroethane.
SnCl4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be exchanged. It is advantageous to use SnCl in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be exchanged most advantageously in an amount from 2.5 to 4.5 moles.
The ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound of formula 1 to be used in excess ranging from 1 to 8. An excess of the compound of formula 1 makes it possible to achieve the highest possible process efficiency within the shortest time.
The reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture. The reaction is conducted in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of obtaining the target product and the greater the quantity of by-products. It is beneficial to conduct the reaction in temperatures from the range from -80 C to 0 C, and most advantageously - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
The reaction of hydroxyl group blocking involves preparation of a solution of a compound containing a hydroxyl group, and the compound of formula 1, followed by cooling of the mixture to a low temperature. After the cooling procedure, SnCl4 is introduced, whereupon the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the blocking reaction by thin-layer chromatography on silica gel plates, or by high-performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
On completion of the reaction, the product is isolated and purified using known methods. Prior to product isolation it is advantageous to neutralize SnCl4 with a neutralizing agent in an amount of at least 4 molar equivalents of SnCl4 used. Depending on product stability, neutralization can be performed with:
1. solutions of bases, solutions of hydrogen carbonate salts, if the product is stable in basic conditions, it is advantageous to use sodium or potassium hydrogen carbonate;
2. buffers with pH of 7 (± 0.7) .
Following neutralization, it is advantageous to filter the mixture in order to separate solid products, whereupon the product is isolated from the post-reaction mixture through extraction with an aprotic organic solvent and then purified by known methods.
A product of hydroxyl function protectionwith an acetal or acetal ester protecting group is an original compound in which the site of the hydroxyl group is occupied by the group of formula 4
where R2 has the meaning defined above.
In the second aspect, the present invention relates to a method for protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives, consisting of the incorporation of an acetal or acetal ester group. The method according to the invention consists of the reaction between the compound of the general formula 5
where
B represents radicals of nucleobases, particularly uracil, or appropriately protected residuesof adenine, guanine, cytosine, uracil and thymine,
Yi and Y2 are the same or different, and represent groups protecting hydroxyl functions in positions 3' and 5 '; in particular, they are silyl groups: triethylsilyl, tert-butyl-dimethyl- silyl, isopropyl-dimethyl-silyl, tert-butyl-diphenyl-silyl, methyl-diisopropyl-silyl, triphenylsilyl, triisopropylsilyl, methyl-di-tert-butylsilyl
or 6
where B has the meaning defined above, and A represents group of formulas 7, 8, 9, 10 and 1 1
and the compound of the general formula 1 (hereinafter referred to as the blocking compound),
R S-CH2-0-R2 (i)
where
- i represents a Cw alkyl; unsubstituted or substituted benzyl or naphthyl, with substituents including a Ci_7 alkyl, halogen, aminoacyl; in particular Ri represents -CH3> -Ph,
-Ph(4-Cl), -Ph(4-CH3), -CH2Ph
- R2 represents
· a Ci_i5 alkyl;
• alkyl-aryl in which the alkyl chain contains Ci_5, and aryl contains from 1 to 8 unsubstituted or substituted rings, with substituents including a Ci_7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
• group of the general formula 2
where:
R3 represents
o a Ci-15 alkyl;
o ketone group
o unsubstituted or substituted phenyl, with substituents including a Ci_7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group,
• group of the general formula 3
where R4, R5 and R<; are different or the same, and represent a Ci_28 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, with the total number of carbon atoms in that group is no less than 6 and no more than 30,
in the presence of (SnCl4), in an aprotic solvent.
According to the invention, the method for the protection of the hydroxyl function in nucleosides consists in particular of incorporation of groups of formulas 12 and 13 in position 2'
The solvents that can be used include halogen derivatives of alkanes, in particular carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane; aromatic solvents: particularly benzene, toluene; cyclic ethers, particularly tetrahydrofuran; nitrile compounds, particularly acetonitrile, or a mixture of these solvents. It is particularly advantageous to use 1,2- dichloroethane.
The reaction is conducted in anhydrous media but it is possible for the reaction to be conducted in an environment containing trace amounts of water, however if this is the case, an appropriate excess of SnCl4 must be ensured because a part of it is bound by water.
In the method according to the invention, the compound of the general formula 5 or 6, in which substituents have the meaning defined above, and an appropriate blocking compound are dissolved in a solvent, and then SnCl4 is added to the reaction mixture. It is advantageous to introduce SnCl4 in the form of a solution in the same solvent as that is the reaction medium or in 1 ,2-dichloroethane.
SnCl4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be protected. It is advantageous to use SnC in an amount from 1 to 6 moles per one mole of hydroxyl groups which are intended to be protected, most advantageously in an amount from 2.5 to 4.5 moles.
The ratio of the compound of formula 1 to hydroxyl groups can be 1 : 1, however it is advantageous for the compound to be used in excess ranging from 1 to 8. An excess of the compound of the formula 1 makes it possible to achieve the highest possible process efficiency withm the shortest time.
The reaction can be conducted over a broad temperature range, from very low temperatures up to temperatures not exceeding the boiling temperature of the reaction mixture, however the temperature of the reaction must not be higher than the temperature of de-protecting the hydroxyl function in positions 3 ' and 5 '. It is advantageous to conduct the reaction in low temperatures, however not lower than the solidification point of the solvent used. The higher the temperature, the lower the efficiency of the process and the greater the quantity of by-products. It is advantageous to conduct the reaction in the temperature range from -80 C to 0 C, and most advantageous - below 15 C. Due to that fact, it is advantageous to use solvents with low solidification points.
The reaction of hydroxyl group protection involves preparation of a solution of a compound containing a hydroxyl group which is to be protected, and a blocking compound, followed by the cooling of the mixture to a low temperature. After the cooling procedure, SnCl4 is introduced, and then the reaction is advantageously conducted at a low temperature until the completion of the process. In order to determine the optimum duration of the process, it is advantageous to monitor the course of the reaction by thin-layer chromatography on silica gel plates, or by high- performance liquid chromatography (HPLC). The duration of the reaction depends on the types of substrates and temperature, and generally ranges from 4 to 16 hours.
On completion of the reaction, the product is isolated and purified using known methods, following the same procedure as in the method according to the first aspect of the invention.
The product of the reaction is the compound of the general formula 14,
where B, R2, Yi and Y2 have the meanings defmed above,
or of the formula 15
where A, B and R2 have the meanings defmed above.
The protecting groups used in the method according to the invention are unblocked in different conditions, on a case-by-case basis, depending on the structure of the group.
A characteristic property of acetal and acetal ester groups protecting the hydroxyl function is high flexibility in unblocking methods. In principle, all known chemical unblocking methods can be employed, however due to the specific properties of acetal and acetal ester groups stemming from the nature of these compounds, it is possible to select appropriate conditions which are advantageous for a particular application of the compounds protected with these groups. The selection of the most advantageous method of unblocking the hydroxyl function protected with the method according to the invention depends not only on the chemical nature of the blocking group but also on the specific properties of the blockage used.
The removal of acetal and acetal ester groups can be performed with solutions of inorganic bases, e.g. NaOH, KOH, and organic bases, e.g. amines in organic or inorganic solvents, also in their mixtures. It is advantageous to use weak bases (e.g. aqueous ammonia solution, methanol/ammonia solution, ethanol/ammonia solution, methylamine in methanol, n-butylamine in methanol), which is why during the unblocking there is practically no hydrolysis of mtemucleotide linkages.
Acetal and acetal ester protecting groups can also be unblocked in acidic conditions, and they are stable in conditions required for the removal of the acid-labile dimethoxytrityl group (DMTr) from the 5 '-hydroxyl position. The groups are stable, which is their advantage, towards weak acid solutions used for the unblocking of the 5 '-hydroxyl group blocked with the acid-labile dimethoxytrityl group (DMTr). In these conditions, the acetal or acetal ester group protecting the 2'-hydroxyl function is stable, which is a significant factor for the chemical synthesis of the RNA chain.
Acetal and acetal ester protecting groups can also be removed in reactions that are specific to a particular blocking group, e.g. in a reaction between the carbonyl group of the ieto-ketoester radical (e.g. levulinyl, Lv, H3CC(0)CH2CH2C(0)-,) and the unblocking reagent i.e. hydrazine solution.
In the third aspect, the subjects of the present invention are new monothioacetals of the general formula 1,
R S-CH2-0-R2 (i)
where
• i represents (4-chloro)phenyl
· R2 represents o-toluyl, benzoyl, pivaloyl.
New monothioacetals are obtained in a reaction between an appropriate chloride of the general formula 16
where R2 has the meanings defined above
and an appropriate derivative of phenylthiomethanol of the general formula 17
/S^/OH
R l ( 17)
where Ri has the meaning defined above,
in an aprotic solvent, advantageously in diethyl ether in the presence of an amine.
As an example, the reaction is conducted in the following manner: 1 eqval of an appropriate compound of formula 17 is dissolved in diethyl ether and combined with 1 egual of an amine. The solution is cooled down to the temperature of 0 C and, on stirring, 1 egualof an appropriate compound of formula 16 is added. On completion of the reaction, the cooling is stopped and a saturated sodium hydrogen carbonate solution is added successively until carbon dioxide no longer evolves from the reaction mixture. The mixture is separated and the organic layer containing the reaction product is dried, following which the solvent is evaporated and the final product is crystallized.
The method according to the invention is universal and can be applied for the protection of hydroxyl functions with acetal and acetal ester groups. The method can be applied for the protection of hydroxyl groups not only in nucleosides and their analogues, but also in alcohols and complex chemical compounds containing a hydroxyl group.
The procedure of incorporating acetal and acetal ester protecting groups into the hydroxyl function is:
• simple - requires the mixing of two reagents and an addition of SnCl4, · economical - uses reagents which are either inexpensive or easy to obtain.
The method makes use of both known and new compounds, developed specifically for the invention, containing a thioacetal or thioacetal ester group.
The method according to the invention has a particularly advantageous application in the chemical synthesis of RNA and its analogues.
The blockage of the hydroxyl function using the method according to the invention is compatible with other protecting groups applied during RNA chain synthesis; for example, it is stable in the conditions of unblocking of the 3'- and 5' -hydroxyl positions with fluoride ions.
The subject of the invention is presented in the following examples which illustrate the invention but do not limit its scope.
Example 1
In a round-bottom flask, 0.2 g (0.41 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl) was dissolved in 3 ml of dry 1 , 2-dichloroethane, and then 0.9 g (3.29 mmol) of anhydrous
benzoyloxymethylthio(4-chloro)benzene was added. To the solution 0.2 g of 4A molecular sieves were added to dry the solution. The flask was closed with a septum provided with argon-filled balloons, and was placed in a cooling bath and cooled down to -25°C; the solution was stirred magnetically. Using a syringe, 1.2 ml of 1.2 M tin(IV) chloride solution (1.43 mmol) in 1,2- dichloroethane were then added. The mixture was stirred for 5 hours, maintaining the temperature of -23°C. The course of the reaction was monitored by TLC (hexane-ethyl acetate-methanol 9:4: 1). On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added to neutralize tin(IV) chloride until bubbles of gas (carbon dioxide) stopped forming, then the cooling was stopped. Following complete neutralization, the reaction mixture was filtered from white precipitate, and then the raw product was extracted three times from the filtrate with 1,2- dichloroethane (3x8ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate, following which the solvent was evaporated. The raw product was purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (99: 1) as eluents. In this manner, 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)- 2'-0-benzoyloxymethyl-uridine was obtained with 89% yield.
Spectroscopic analysis:
lH NMR (400MHz, CDC13) 0,893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2,4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2 ' , H-3 \ m), 4.378 (1H, H-5 ', d,
J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6,4Hz), 5.784-5.800 (2H, OCH20, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5,6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m)
13C NMR (400MHz, CDC13) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH3), 59.25 (C-5'), 67.53 (C-3'), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20), 89.33
(C-l'), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)
Example 2
Following the procedure defined in Example 1, a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 1.665 g (6 mmol) of benzoyloxymethylthio(4-chloro)benzene and 2.3 ml of 1 M tin(IV) chloride
(2.5 mmol) in 1,2-dichloroethane. The reaction was conducted over a 24-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was added until bubbles of gas (carbon dioxide) stopped forming. In this manner, 3',5'-0-(tetraisopropyldisiloxane- l,3-diyl)-2'-0-benzoyloxymethyluridine was obtained with 82% yield. NMR analysis confirmed the structure of the product.
Example 3
Following the procedure defined in Example l,a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.57 g (2 mmol) of benzoyloxymethylthio(4-chloro)benzeneand 1.180 ml of 1.3 M tin(IV) chloride solution (1.54 mmol) in 1,2-dichloroethane. The reaction was conducted over a 48-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, S'^'-O-itetraisopropyldisiloxane-l^-diyl^'-O- benzoyloxymethyl-uridine was obtained in the yield of 15%. NMR analysis confirmed the structure of the product.
Example 4
Following the procedure defined in Example 1, a reaction was conducted in 2 ml of 1,2- dichloroethane between 0.2 g (0.3 mmol) of N6-phenoxyacetyl-3',5'-0-(tetraisopropyldisiloxane- l,3-diyl)adenosine and 0.69 g (2 mmol) of benzoyloxymethylthio(4-chloro)benzene and 1.120 ml of 0.9 M tin(IV) chloride solution (1.05 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, N6-phenoxacetyl-2'- benzoyloxymethyl-3',5'-(tetraisopropyldisiloxane-l,3-diyl)adenosine was obtained in the yield of 72%.
Spectroscopic analysis:
Ή NMR (400MHz, CDC13) 9.50 (s, 1H, NH); 8.72 (s, 1H, H-2); 8.25 (s, 1H, H-8); 8.071-8.107 (2H, H-Ar, m) 7.44-6.81 (m, 7H, H -Ar,H-Ar-Pac); 6.22 (d, J = 4.7 Hz, 1H, H-l'); 5.51-5.40 (2H, m OCH20); 5.08 (t, 3JH2' H3',H1' = 4.7 Hz; 1H, H-2 ' ) ; 4.88 (s, 2H, NHCOC¾Ph); 4.55 (q, 3jH37H2',OH3 ',H4'= 4.7 Hz, 1H, H-3 '); 4.28 (m, 1H, H-4'); 3.54 (dd„lH, H-5'); 3.43 (dd, 2JH5"/H5' = 10.7 Hz; 3JH5" H4'= 4 Hz, 1H, H5"); 2.75 (1H, m); 0,893-1.089 (28H, m).
13CNMR (400MHz, CDCI3) 166.7 (NHCO);165.83 (C=0); 158.6 (C); 157.2 (C, Pac); 152.6 (C2); 151.5 (C6); 148.4 (C4); 144.4, 135.5 (C, Car); 142.2 (C8); 130.1, 129.9, 128.1, 127.9122.4, 115, 114.9, 113.2 (12H, Ar); 123.2 (C5); 89.0 (OCH20); 87.3 (CI'); 84.2 (C4'); 81.7 (C2'); 70.5 (C3'); 68.1
Example 5
Following the procedure defined in Examplel, a reaction was conducted in 6 ml of 1,2- dichloroethane between 0.2 g (0.27 mmol) o f N2-tert-butylphenoxyacetyl-3',5'-0- (tetraisopropyldisiloxane-l,3-diyl)guanosine and 0.62 g (2.2 mmol) of benzoyloxymethylthio(4- chloro)benzene and 1.2 ml of 067 M tin(IV) chloride solution (0.81 mmol) in dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, N2- tert-butylphenoxacetyl-3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- benzoyloxymethylguanosine was obtained in the yield of 74%.
Spectroscopic analysis:
¾NMR (400MHz, CDC13) 11.78 (s, 1H, NH-1); 9.09 (s, 1H, NHPac); 7.79 (s, 1H, H-8); 7.36-6.73 (m, 9H, H-Ar, H-tbuPac); 5.99 (H, H-l'); 5.46-5.36 (2H, m, OCH20); 5.23 (s, 2H, NHCOCH2Ph); 4.65 (t,
1H, H-2'); 4.34 (m, 1H, H-3'); 4.17 (m, 1H, H-4'); 3.38 (m,lH, H-5'); 3.35 (dd, 2JH5"/H5' = 10.7 Hz; 3JH5"/H4'= 2.9 Hz, 1H, H-5"); 2.49 (d, J= 4.3 Ηζ,ΙΗ, OH3'); 1.15 (s, 9H, tbuPac), 0.893-1.089 (28H, m).
^NMR (400MHz, CDC13) 169.9 (NHCO); 165.83 (C=0), 158.6 (C, Car); 155.2 (C6); 154.2 (4C, tbuPac);
147.7 (C2); 146.5 (C4); 145.9 (C, tbuPac); 144.3, 135.5 (C, ); 137.0 (C8); 130.1, 128.1, 127.1
126.9, 126.8, 114.4, 113.3 (CH, Car); 122.3 (C5); 89.3 (OCH20); 86.1 (CI'); 84.0 (C2'); 82.7 (C4'); 70.4 (C3'); 67.1 (NHCOCH2Ph); 63.1 (C5'); 34.3 (C, PhC(CH3)3); 31.4 (PhC(CH3)3), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (GH3)3,12.45, 12.81, 12.99, 13.23 (CH). Example 6
Following the procedure defined in Examplel, a reaction was conducted in 1 ml of 1,2- di chloroethane b etwe en 0.05 g ( 0. 1 mmo l) o f 3 ' , 5 '-0- (tetraisopropyldisiloxtetraisopropyldisiloxane-l,3-diyl)uridine and 0.266 g (1 mmol) of benzoyloxymethylthio(4-methyl)benzene and 0.45 ml of 1 M tin(IV) chloride (0.45 mmol) in dichloroethane. The reaction was conducted over a 4.5-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped
evolving. In this manner, 3 ',5'-(tetraisopropyldisiloxane-l,3-diyl)-2'-0-(benzoyloxymethyl)undine was obtained in the yield of 85%/
Spectroscopic analysis:
lR NMR (400MHz, CDC13) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4\ J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz),
5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l \ d, J=6.4Hz), 5.784-5.800 (2H, OCH20, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) 13C NMR (400MHz, CDC13) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH3)3, 59.25 (C-5 '), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20), 89.33 (C-1 ' 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83
(C=0)
Example 7
Following the procedure defined in Example 1, a reaction was conducted in 1 ml of 1,2- dichloroethane between 0.05 g (0. 1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.244 g ( 1 mmol) of benzoyloxymethylthiobenzeneand 0.45 ml of 1 M tin(IV) chloride (0.45 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 8-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetra-isopropylsiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 80%.
Spectroscopic analysis:
lR NMR (400MHz, CDC13) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2,4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l \ d, J=6.4Hz), 5.784-5.800 (2H, OC¾0, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) 13C NMR (400MHz, CDC13) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH3), 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20), 89.33 (C- 1 '), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (CO) Example 8
Following the procedure defined in Example 1, a reaction was conducted in 5 ml of 1,2- dichloroethane between 0.5 g (1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 2
g (1 mmol) of toluyloxymethylthio(4-chloro)benzeneand 2.40 ml of 1.25 M tin(IV) chloride (3 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 12-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (o-toluyloxymethyl)uridine was obtained in the yield of 85 %.
Spectroscopic analysis:
lH NMR (400MHz, CDC13) 0.893-1.089 (28H, m), 2.615 (3H, s, CH3), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz), 5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6.4Hz), 5.784-5.800 (2H, OCH20, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107
(2H, H-Ar, m)
13C NMR (400MHz, CDC13) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.3 1, 17.34, 17.41 (CH3), 21 .72 (CH3), 59.25 (C-5 '), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH2O), 89.33 (C-l '), 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)
Example 9
Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.5 g ( 1 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 1.58 g (6 mmol) of pivaloyloxymethylthio(4-chloro)benzene and 2.3 ml of 1 M tin(IV) chloride solution (2.55 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)uridine was obtained in the yield of 75%.
Spectroscopic analysis:
Ή NMR (400MHz, CDC13) 0.955-1. 102 (28H, m), 1.230 (9H, (C/¾3, s), 3.951, 3.985 (1H, H-5", dd, J=2Hz, J=2Hz), 4.113, 4.137 (1H, H-4 ', dd, J= 1.6Hz, J= 1.6Hz), 4.21-4.303 (3H, H-2',3',5', m), 5.496, 5.561 (2H, (X¾0, dd, J=6.4Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.743 (1H, H-l ', s), 7.860 (1H, H-6, d, J=8Hz)
13C NMR (400MHz, CDC13) 12.55, 12.84, 13.06, 13.39 (CH), 16.80, 1691, 16.95, 17.10, 17.21, 17.28, 17.38, 17.46 (CH3), 26.97 ((CH3)3), 38.79 (C(CH3)3), 59.30 (C-5'), 67.99 (C-3'), 81.27 (C-2'), 81.61 (C-4'),
87.76 (C-1 ' 89.06 (OCH20), 101.53 (C-5), 139.39 (C-6), 149.50 (C-2), 193.03 (C-4), 178.03 (CO) Example 10
Following the procedure defined in Example 1, a reaction was conducted in 2 ml of 1,2- dichloroethane between 0.2 g (0.4 mmol) of 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)undine and 0.97 g (4 mmol) of pivaloyloxymethylthio(4-methyl)benzene and 2 ml of 0.9 M tin(IV) chloride solution (1.8 mmol) in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)uridine was obtained in the yield of 75%.
Spectroscopic analysis:
lR NMR (400MHz, CDC13) 0.955-1.102 (28H, m), 1.230 (9H, (C¾)3, s), 3.951, 3.985 (1H, H-5", dd, J=2Hz, J=2Hz), 4.113, 4.137 (1H, H-4 ', dd, J= 1.6Hz, J= 1.6Hz), 4.21-4.303 (3H, H-2',3',5', m), 5.496, 5.561 (2H, OCi¾0, dd, J=6.4Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.743 (1H, H-l\ s), 7.860 (1H, H-6, d, J=8Hz)
13C NMR (400MHz, CDC13) 12.55, 12.84, 13.06, 13.39 (CH), 16.80, 1691, 16.95, 17.10, 17.21, 17.28, 17.38, 17.46 (C¾), 26.97 ((CH3)3), 38.79 (C(CH3)3), 59.30 (C-5'), 67.99 (C-3'), 81.27 (C-2'), 81.61 (C-4'), 87.76 (C-Γ), 89.06 (OCH20), 101.53 (C-5), 139.39 (C-6), 149.50 (C-2), 193.03 (C-4), 178.03 (C=0) Example 1 1
Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.25 g (0.51 mmol) of 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine and 0.86 g (5.1 mmol) of benzoyloxymethylthiomethyl ether and 2.3 ml of 1 M tin(IV) chloride (2.3 mmol) solution in 1,2-dichloroethane. The reaction was conducted over an 8-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3',5'-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 80%.
Spectroscopic analysis:
lR NMR (400MHz, CDC13) 0.981-1.110(m, 28H), 3.977, 4.011 (1H, H-5', dd, J=2.4 Hz, J=2.4Hz,), 4.150, 4.173 (1H., H-4 ' , dd, J=2Hz, J=2Hz,), 4.229-4.283 (3H, H-2' ,3 ',5 " , m,) 4.776, 4.702 (2H, OC¾C6H5 dd, J=12Hz, J=12Hz), 4.967, 5.057 (2H, OC¾0, dd, J=6.8Hz, J=6.8Hz), 5.668 (1H, H-5, d, J=8Hz), 5.803 (1H, H-l ', s), 7.311-7.374 (5h, H-Ar, m), 7.885 (1H, H-6, d, J=8Hz)
13C NMR (400MHz, CDC13) 12.58, 12.86, 13.09, 13.37 (CH), 16.83, 16.94, 16.98, 17.06, 17.21, 17.31, 17.39, 17.48 (CH3), 59.36 (C-5'), 68.03 (OCH2Ar), 69.17 (C-3 '), 77.99 (C-2'), 81.83 (C-4'), 89.34 (C-1 ' 93.07 (OCH2O),101.902 (C-5') 127.63-128.32 (5C-Ar), 137.51 (lC-Ar), 139.34 (C-6), 149.64 (C-2), 163.01 (C-4)
Example 12
Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.41 mmol) of 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)undine and 0.728 g (4.1 mmol) of benzoyloxymethylthiomethyl ether and 1.7 ml of 1 M tin(IV) chloride ( 1 8 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 12-hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 3 ',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (benzoyloxymethyl)uridine was obtained in the yield of 85%.
Spectroscopic analysis:
lR NMR (400MHz, CDC13) 0.893-1.089 (28H, m), 3.951, 3.985 (1H, H-5", dd, J=2.4Hz, J=2.4Hz), 4.149, 4.173 (1H, H-4', J=1.6Hz, J=1.6Hz), 4.201-4.264 (2H, H-2', H-3 ', m), 4.378 (1H, H-5', d, J=4.4Hz),
5.68 (1H, H-5, d, J=8Hz), 5.758 (1H, H-l ', d, J=6.4Hz), 5.784-5.800 (2H, OCH20, m) 7.407-7.470 (2H, H-Ar, m), 7.561 (1H, H-Ar, t, J=5.6), 7.867 (1H, H-6, d, J=8Hz), 8.071-8.107 (2H, H-Ar, m) 13C NMR (400MHz, CDC13) 12.45, 12.81, 12.99, 13.23 (CH), 16.70, 16.84, 16.92, 17.18, 17.24, 17.31, 17.34, 17.41 (CH3), 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20), 89.33 (C- 1 '), 101.58 (C-5), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0)
Example 13
Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.52 mmol) of 3 ' ,5 '-di-fert-butyl-silylene-uridine and 1 g (4.2 mmol) of anhydrous triisopropylsilyl(ethylthio)methyl ether and 1.7 ml of 1 M anhydrous tin(IV) chloride ( 1.82 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was
added until bubbles of gas stopped forming. In this manner, 3 ',5'-0-(di-tert-butyl-silylene)-2'-0- [[(triisopropylsilyl)oxy]methyl]uridine was obtained in the yield of 75%.
lH NMR (400MHz, CDC13) δ (ppm): 1.12 (m, 39H), 3.965 (lH,m), 4.152 (1H, H-4'), 4.241 (2H, H-2', H-3\ m), 4.364 (1H, H-5'), 5.62 (1H, d, J=8Hz), 5.732 (1H, d, J=6.4Hz), 5.796 (2H, OCH20, m) 7.867 (1H, d, J=8Hz)
13C NMR (400MHz, CDC13) δ (ppm): 11.98, 12.02, 12.05, ((CH3)2CH), 16.89, 17.08, 17.19, 17.24, 17.31, 17.36 (CH3), 27.04, 27.09, 27.13, 27.28 27.35, 27.62 (CH3, f-butyl), 32.65, 32.76(CH, /-butyl) 58.98 (C-5'), 67.13 (C-3'X 81.56 (C-2'), 82.32 (C-4'), 88.56 (OCH20), 89.17 (C-l'), 101.24 (C-5), 138.53 (C-6), 148.79 (C-2), 162.96 (C-4)
Example 14
Following the procedure defined in Example 1, a reaction was conducted in 3 ml of 1,2- dichloroethane between 0.2 g (0.48 mmol) of 3 ',5'-di-ier/-butoxysilylene-uridine and 1.12 g (3.8 mmol) of anhydrous (p-chlorophenylthiometoxy)-ferf-butyldimethylsilane and 1.7 ml of 1 M anhydrous tin(IV) chloride (1.68 mmol) solution in 1,2-dichloroethane. The reaction was conducted over a 6-hour period. On completion of the reaction, a saturated aqueous solution of hydrogen carbonate was added until bubbles of gas stopped forming. In this manner, 3',5'-0-(di-teri-butoxy- silylene)-2'-0-[[(di-methyl-teri-butylsilyl)oxy]methyl]-uridine was obtained in the yield of 73%. ¾ NMR (400MHz, CDC13) δ (ppm): 0.16 (m, 6H), 1.02 (m, 9H), 1.35 (m, 18H), 3.965 (lH,m), 4.152 (1H, H-4'), 4.241 (2H, H-2', H-3 ', m), 4.364 (1H, H-5'), 5.62 (1H, d, J=8.2Hz), 5.732 (1H, d, J=6.4Hz),
5.698 (2H, OCHjO, m) 7.867 (1H, d, J=8.2Hz),
13C NMR (400MHz, CDC13) δ (ppm): -4.9 (CH3), 23.09, 23.29, 23.54 (CH3, f-butyl), 31.04 (C, /-butyl), 32.04, 32.09, 32.13, 32.28 32.35, 32.62 ((CH3)3COSi, /-butyl), 58. 64 (C-5'), 68.35 (C-3'), 74.68, 74.79 ((CH3)3COSi, /-butyl), 82.42 (C-2'), 83.06 (C-4'), 88.93 (OCH20), 89.91 (C-l'), 101. 43 (C-5), 138.68 (C-6), 147. 59 (C-2), 163.33 (C-4)
Example 15
A volume of 30 μΐ (0.3 mmol) of dry benzyl alcohol was transferred into a round-bottom flask and then dissolved in 1 ml of dry 1,2-dichloroethane, then 0.166 g (0.6 mmol) of benzoyloxymethylthiobenzene was added in the presence of 4 A molecular sieves. The flask was closed with a septum provided with an argon-filled balloon. The mixture was cooled down to the temperature of -25°C and thereafter, on stirring, 0.6 ml of 0.8 M solution of tin(IV) chloride (0.48
mmol) in 1,2-dichloroethane was added. The reaction was conducted in argon atmosphere. The mixture was stirred magnetically at a temperature of -25°C for 5 hours. Thereafter, the reaction was completed by adding an aqueous solution of sodium hydrogen carbonate until carbon dioxide stopped evolving,then the cooling bath was removed. The white precipitatewas filtered off, and the filtrate was extracted three times with 1,2-dichloroethane (3x3 ml). The organic layers were collected and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The raw product was purified on a preparative PLC plate covered with silica gel 60 RP-18, F254, 1 mm, from Merck, using hexane-dichloromethane 2:3 as the mobile phase. The product was extracted with dichloromethane (15 ml). In this manner, benzyloxymethylbenzoyl was obtained in the yield of 56%.
Spectroscopic analysis:
Ή NMR (400MHz, CDC13) 4.8 (2H, s, CH2), 5.6 (2H, s, OCH20), 7.35-7.679 (8H, m), 8.02 (2H, d, J=lHz) 13C NMR (400MHz, CDC13) 68.02 (1C, C¾), 98.58 (1C, OC¾0), 136.21 (lC-Ar), 126.68-133.02 (11C- Ar), 166.57 (C=0)
Example 16
A portion of 0 .25 0 g ( 0 .4 m m o l ) o f 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- benzoyloxymethyluridine obtained according to Example 1 was dissolved in 6 ml of anhydrous tetrahydrofurane (THF), then 0.5 ml of 1 Mtriethylammonium fluoride in THF was added. The mixture was stirred for 18 hours at room temperature. The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted six times with 5 ml of methylene dichloride . The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The product, 2'-0-benzoyloxymethyl- uridine, was purified in a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (96:4) as eluents. The yield was 82%.
Spectroscopic analysis:
lR NMR (400MHz, DMSO-de) 3.546-3.596(m, 1H), 3.60-3.678 (m, 1H), 3.877-3.902 (m, 1H), 4.157-4.227 (m, 1H), 4.405(t, 1H), 5.165 (t, lH), 5.323 (d, 1H, J=5.6 Hz), 5.527 (d, 1H, J=6.4Hz), 5.596 (t, 1H,
J=8Hz), 5.937 (t, 1H, J=5.2Hz), 7.666 (t, lH-arom, J=7.2Hz), 7.912-8.057 (m, 3H-arom), 7.491-7.583
(2H, m, lH-arom, 1H-6)
13C NMR (400MHz, DMSO-d6) 59.25 (C-5'), 67.53 (C-3'), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20),
89.33 (C-1' 101.58 (C-5), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0).
Example 17
A portion of 50 mg (0.13 mmol) of 2 '-0-benzoyloxym ethyl -uridine obtained in Example 16 was dissolved in 2.5 ml of THF, then2.5 ml of 2 M solution of n-butylamine in methanol was added. On completion of the unblocking reaction (21 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μπι) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis.
Spectroscopic analysis:
¾ NMR (400MHz, DMSO-d6) 3.516-3.567 (lH,m); 3.567-3.644 (lH,m); 3.823-3.849 (lH,q, J=3.6Hz, J=7.2Hz), 3.939-3.974 (1H, q, J=5.2Hz, 9.2Hz), 3.999-4.039 (1H, q, J=5.6Hz, J=10.8Hz), 5.060-5.089
(2H,m), 5.358 (1H, d, J=5.5Hz), 5.629 (1H, d, J=4Hz, H-5), 5.776 (1H, d, J=5.6Hz, H-l'), 7.880 (1H, d, J=4Hz, H-6), 11.297 (lH,s, ΝΉ)
"C NMR (400MHz, DMSO-d6) 60.85(C-5'); 69.88(C-3 ') ; 73.54(C-2') ; 84.83 (C-4'); 87.68(C-1') ;
101.75(C-5) ; 140.72(C-6); 150.74(C-2); 163.12(C-4)
Example 18
A portion of 0.250 g ( 0.4 16 mmol) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- pivaloyloxymethyluridine obtained according to Example 9 was dissolved in 6 ml of dry tetrahydrofurane (THF), whereupon 0.5 ml of 1 M triethylammonium fluoride in THF was added. The mixture was stirred for 18 hours in room temperature . The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted six times with 5 ml of methylene di chloride. The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product of the reaction was
purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (95 :5) as eluents. In this manner, 2'-0-(pivaloyloxymethyl)uridine was obtained in the yield of 84%.
Spectroscopic analysis:
Ή NMR (400MHz, DMSO-dg) 3.546-3.596(m, 1H), 3.60-3.678 (m, 1H), 3.877-3.902 (m, 1H), 4.157-4.227 (m, 1H), 4.405(t, 1H), 5.165 (t, lH), 5.323 (d, 1H, J=5.6 Hz), 5.527 (d, 1H, J=6.4Hz), 5.596 (t, 1H, J=8Hz), 5.937 (t, 1H, J=5.2Hz), 7.491-7.583 (1H, d, H-6, J=6.4 Hz)
13C NMR (400MHz, DMSO-d6) 59.25 (C-5'), 67.53 (C-3 '), 81.72 (C-2'), 82.63 (C-4'), 88.42 (OCH20), 89.33 (C-1 ' 101.58 (C-5'), 128.27-133.25 (6C-Ar), 139.51 (C-6), 149.79 (C-2), 163.47 (C-4), 165.83 (C=0).
Example 19
A portion of 50 mg (0.14 mmol) of 2'-(9-(pivaloyloxymethyl)uridine obtained in Example 18 was dissolved in THF (2.5 ml). Thereafter, 2 M n-butylamine solution in methanol (2.5 ml) was added. On completion of the unblocking reaction (34 hours), the solvent and amine residue were evaporated from the reaction mixture. The post-reaction mixture was introduced into a chromatography column packed with silica gel 60 (63-200 μηι) from Merck, using methylene dichloride-methanol (60:40) as eluents. Uridine was isolated as the product of unblocking. NRM analysis confirmed that the compound resulting from the removal of the protecting group is uridine. Example 20
100 mg (0.16 mmol) of 3',5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0-(benzoyloxymethyl)- uridine obtained in Example 1 was dissolved in 2.5 ml of THF . Thereafter, 2.5 ml of 2 M n- butylamine solution in methanol was added. On completion of the unblocking reaction (48 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (99: 1) as eluents. The isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine.
Spectroscopic analysis:
lR NMR (400MHz, CDC14)0.982-1.096 (28H, m), 3.339 (1H, d, J=2Hz), 3.985, 4.019 (1H, dd, J=2.8Hz, J=2.8Hz), 4.115-4.141 (1H, m), 4.187-4.221 (2H, m), 4.327-4.361 (1H, m), 5.695 (1H, d, J=8Hz, H- 5), 5.734 (1H, s, H-l'), 7.720 (1H, d, J=8Hz, H-6)
13C NMR (400MHz, CDC14) 12.46-13.34 (CH, 4C), 16.78-17.42 ( CH3> 8Q60.23 (C-5'), 68.89 (C-3'), 75.14 (C-2'), 81.90 (C-4'), 90.91 (C-l '), 101.94 (C-5'), 139.95 (C-6), 150.00 (C-2), 163.26 (C-4).
Example 21
A portion of 96 mg ( 0 . 1 6 mm o l) o f 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- (pivaloyloxymethyl)-uridine obtained in Example 10 was dissolved in THF (2.5 ml). Thereafter, 2.5 ml of 2 M n-butylamine solution in methanol was added. On completion of the unblocking reaction (52 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μπι) from Merck, using methylene dichloride-methanol (98 :2) as eluents. The isolated product of unblocking of the protecting group in position 2 ' is 3 ' ,5 '-0-(tetraisopropyldisiloxane-l,3- diyl)uridine. Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
Example 22
A portion of 1 02 mg (0 . 1 6 mmo l) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3-diyl)-2'-0- toluyloxymethyluridine obtained in Example 8 was dissolved in 2.5 ml of THF, then 2.5 ml of 2 M solution of n-butylamine in methanol was added. On completion of the unblocking reaction (72 hours), the solvent and amine residue were evaporated from the reaction mixture. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μηι) from Merck, using methylene dichloride-methanol (98:2) as eluents. The isolated product of unblocking of the protecting group in position 2' is 3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)uridine. Spectroscopic analysis confirmed the identification of the compound - cf. the spectrum in Example 20.
Example 23
A portion of 42 μΐ ( 1 .6 mmol) of hydrofluoric acid-pyridine complex (HF-Py, Aldrich) was carefully added, while cooling, to 0.2 ml of pyridine. To the solution at a temperature of 0°C, upon magnetic stirring, 0 . 2 3 0 g ( 0 . 4 m m o l ) o f 3 ' , 5 '-0-(di-tert-butyl-silylene)-2'-0- [[(triisopropylsilyl)oxy]methyl]uridine obtained in Example 13 was added in 6 ml of anhydrous
methylene dichlonde. The mixture was stirred for 2 hours at a temperature of 0°C. The course of the reaction was monitored by TLC (methylene chloride-methanol 9: 1 v/v). On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming. The product was extracted with dichloromethane several times (6x5 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The product, 2'-0- [[(triisopropylsilyl)oxy]methyl]uridine, was purified in a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (96:4) as eluent. In this manner, 2'-0-[[(triisopropylsilyl)oxy]methyl]uridine was obtained in the yield of 76%.
Ή NMR (400MHz, CDC13) δ (ppm): 1.19 (21H,m), 3.784 (m, 1H), 4.049 (m, 1H), 4.075 (m, 1H), 4.234(t, 1H, J=4Hz), 4.364 (t,lH, J=4Hz), 5.518(m, 2H, OCH20), 5.698 (d, 1H, J=8Hz), 5.713 (d, 1H, J=3.6Hz), 7.736 (1H, d, H-6, J=8Hz)
13C NMR (400MHz, CDC13) δ (ppm): 12.23, 12.29, 12.34 (CH), 17.54, 17.62, 17.69, 17.86, 18.06, 18.12 (CH3), 57.96 (C-5'), 68.23 (C-3'), 82.56 (C-2'), 83.24 (C-4'), 89.32 (C-l'), 90.56 (OCH20), 101.34 (C-5), 139.53 (C-6), 149.92 (C-2), 163.76 (C-4)
Example 24
Following the procedure defined in Example23, a reaction was conducted by carefully adding 42 μΐ (1.6 mmol) of hydrofluoric acid-pyridine complex (HF-Py, Aldrich), on cooling, to 0.2 ml of pyridine. To the solution at a temperature of 0°C, a portion of 0.220 g (0.4 mmol) of 3',5'-0-(di- tert-butoxy-silylene)-2'-0-[[(di-methyl-t-butylsilyl)oxy]methyl]uridine in 6 ml of anhydrous methylene dichloride was added at a temperature of 0°C. The reaction was conducted for 2 hours at a temperature of 0°C. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this manner, 2'-0-[[(di-methyl-t- butylsilyl)oxy]methyl]uridine was obtained in the yield of 74%.
Ή NMR (400MHz, CDC13) δ (ppm): 0.96 (m, 6H) 1.106 (m, 9H) 3.794 (m, 1H), 4.0 1 (m, 1H), 4.089 (m, 1H), 4.296 (t, 1H, J=4Hz), 4.384 (t,lH, J=4Hz), 5.496 (m, 2H, OCH20), 5.726 (d, 1H, J=8Hz), 5.763 (d, 1H, J=3.6Hz), 7.724(1H, d, H-6, J=8Hz)
13C NMR (400MHz, CDC13) δ (ppm): -4.8 (CH3), 19.78 (C, /-butyl) 23.09, 23.29, 23.54 (CH3, /-butyl), 57.46 (C-5'), 68.29 (C-3'), 82.56 (C-2'), 83.14 (C-4'), 88.97 (OC¾0), 89.94 (C-l'), 102.34 (C-5), 139.68 (C-6), 148.925 (C-2), 164.23 (C-4)
Example 25
A portion of 0. 108 g (0.25 mmol) of 2 '-0-[[(triisopropylsilyl)oxy]methyl]uridine obtained in Example 23 was dissolved in 5 ml of THF, then 0.3 ml of 1 Mtetrabutylammonium fluoride in THF was added. On completion of the unblocking reaction (5 hours), a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming.The product was extracted with dichloromethane several times (6x10 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product was introduced into a chromatographic column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.
Example 26
A portion of 0.1 g (0.25 mmol) of 2'-0-[[(di-methyl-t-butylsilyl)oxy]methyl]uridine obtained in Example 24 was dissolved in 5 ml of THF, then 0.3 ml of 1 Mtetrabutylammonium fluoride in THF was added. On completion of the unblocking reaction (3.5 hours), a saturated solution of sodium hydrogen carbonate was added until bubbles of gas stopped forming.The product was extracted with dichloromethane several times (6x10 ml). The organic layers were dried over anhydrous sodium sulphate. The solvent was evaporated. The raw product was introduced into a chromatography column packed with silica gel 60 (63-200 μιη) from Merck, using methylene dichloride-methanol (60:40) as eluents. The collected fraction was evaporated. The isolated product of the unblocking reaction was uridine, which was confirmed by NMR analysis - cf. the spectrum in Example 17.
Example 27
A portion of 35 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 10 g (0.057 mol) of 4-chlorophenylthiomethanol, whereupon 4.6 ml (0.057 mol) of anhydrous pyridine was added. The solution was placed in a cooling bath with a temperature of 0°C, then on stirring, 8.01 g (0.057 mol) of anhydrous benzoyl chloride was added. The reaction was conducted for 2 hours at a temperature of 0°C, whereupon the cooling bath was removed.
During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate (ca. 60 ml) was added to the reaction mixture until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate wasdissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The product was crystallized from diethyl ether. In this manner, benzoyloxymethylthio-(4-chloro)benzene was obtained in the yield of 89%.
Spectroscopic analysis:
Ή NMR (400MHz, CDC13) 5.634 (2H, s), 7.302-7.317 (2H, H-Ar., m), 7.442-7.475 (4H, H-Ar, m), 7.591
(1H, H-Ar t, J=8), 8.035-8.056 (2H, H-Ar, m )
13C NMR (400MHz, CDC13) 68.66 (-OCH2S-), 127.41, 128.36, 128.43, 128.73, 129.22, 129.40, 129.42,
129.66, 132.04, 133.06, 133.37, 133.67 (C-arom), 165.67 (C=0)
Example 28
A portion of 10 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 3 g (17 mmol) of 4-chlorophenylthiomethanol, whereupon 1.38 ml (17 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, whereupon, onstirring, 2.247 ml (17 mmol) of anhydrous o-toluyl chloride was added. The reaction was conducted for 2 hours at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added to the reaction mixture until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved.The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. In this manner, o-toluyloxymethylthio-(4-chloro)benzene was crystallized from diethyl ether in the yield of 95%.
Spectroscopic analysis:
¾ NMR (400 MHz, CDC13) 2.615 (3H, s, C¾), 5.643 (2H, s, OCH2S), 7.272-7.295 (2H, m, H-Ar), 7.319 (2H, m, H-Ar), 7.321-7.338 (2H, m, H-Ar),7.430-7.449 (1H, m), 7.462-7.479 (2H, m), 7.917-7.937 (1H, m)
13C NMR (400 MHz, CDC13) 21.72 (CH3), 68.28 (OC¾S), 125.79-140.72 (12C-Ar) 166.45 (C=0)
Example 29
A portion of 2 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 0.5 g (2.8 mmol) of 4-chlorophenylthiomethanol (0.5 g; 2.8 mmol; 1 egual), whereupon 0.3 ml (3.65 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, then on stirring, 0.37 ml (3 mmol) of anhydrous pivaloyl chloride was added. The reaction was conducted for 60 min at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate appeared. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. The product was crystallized from diethyl ether. In this manner, pivaloyloxymethylthio-(4-chloro)benzene was obtained in the yield of 78%.
Spectroscopic analysis:
Ή NMR (400 MHz, CDC13) 1.198 (9H, s), 5.373 (2H,s), 7.285-7.302 (2H, m, H-Ar), 7.385-7.402 (2H, m, H- Ar)
13C NMR (400 MHz, CDC13) 26.9 ((CH3)3), 38.78 (C(CH3)3), 67.96 (OCH2S), 129.15, 129.23, 131.71,
131.89, 133.24, 136.11 (C-arom), 177.52 (C=0)
Example 30
A portion 4 ml of anhydrous diethyl ether was transferred into a round-bottom flask and used for dissolving 2 g (12 mmol) of 4-methylphenylthiomethanol, then 0.96 ml (12 mmol) of anhydrous pyridine was added. The mixture was placed in a cooling bath with a temperature of 0°C, then on stirring, 1.6 ml (12 mmol) of anhydrous pivaloyl chloride was added. The reaction was conducted for 60 min at a temperature of 0°C, then the cooling bath was removed. During the reaction, pyridine hydrochloride precipitate was formed. On completion of the reaction, a saturated aqueous solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. The pyridine hydrochloride precipitate was dissolved. The ether layer with the product of the reaction was isolated and dried over anhydrous sodium (VI) sulphate. The solvent was evaporated. In this manner, pivaloyloxymethylthio-(4-methyl)benzene in the form of an oily liquid was obtained in the yield of 63%.
Spectroscopic analysis:
Ή NMR (400MHz, CDC13) 1.198 (9H,s), 2.197 (3H, s), 4.629 (2H, s, (-SCH20-)), 7.335-7.368 (4H, m, H- arom)
13C NMR (400MHz, CDC13) 21.05 (CH3), 26.96 ((CH3)3), 38.79 (C(CH3)3), 69.77 (-SCH20-), 129.78 (2C- arom), 131.06 (C-arom), 131.32 (2C-arom), 137.60 (C-arom), 177.68 (C=0)
Example 31
A portion of 1.36 g (35 mmol) of imidazole was added to a solution of 3 g (17 mmol) of 4- chlorophenylthiomethanol in 20 ml of anhydrous methylene chloride, and cooled down in the c o o l i ng b ath w ith a te m p e ratu re o f 0 °C . The re afte r, 2. 72 g ( 1 8 m m o l ) o f t- butyldimethylchlorosilane was added and the mixture was left to heat to room temperature, then the reaction was conducted for 16 hours. 50 ml of methylene chloride and 50 ml of 5% aqueous solution of NaH2P04 were added to the mixture. The organic layer was dried with anhydrous sodium sulphate and then evaporated under reduced pressure, obtaining an oil of the raw product which was purified by chromatography in a column with silica gel 60 (63-200 μιη) from Merck, using dichloromethane-hexane (85 : 15) as eluent. In this manner, (p-chlorophenylthiomethoxy)-t- butyldimethylsilane in the form of thick oil was obtained in an amount of 3.5 g, in the yield of ca. 70%.
lR NMR (400 MHz, CDC13, ppm): 0.12 (H3CSi, 3H), 0,91 (CH3C, 9H), 5.32 (s, SCH20, 2H), 7.32 (m, 2H, Ar), 7.46 (m, 2H, Ar).
13C NMR (400 MHz, CDC13, ppm): -4.8, 19.32, 25.35,25.72, 25.98, 69.77 (-SCH20-), 129.781, 130.14, 131.06, 131.32, 131.57, 137.60
Example 32
Following the procedure defined in Example 1, a reaction was conducted in 17 ml of 1,2- dichl oroethane b etween 1 g ( 1 .9 mmol) of 3 ' , 5 '-0-(tetraisopropyldisiloxane-l,3- diyl) adenosine and 4.58 g (15.7 mmol) of toluiloxymethylthio(4-chloro)benzene and 1.778 ml of 3,75 M tin(IV) chloride solution (1.05 mmol) in 1,2-dichloroethane. The reaction was conducted over a 24 hour period. On completion of the reaction, a saturated solution of sodium hydrogen carbonate was added until carbon dioxide stopped evolving. In this
manner, 2'-0-toluiloxymethyl-3 ',5'-0-(tetraisopropyldisiloxane-l,3-diyl)adenosine was obtained in the yield of 67%. Spectroscopic analysis:
'H NMR (400MHz, CDC13) 8.918 (s, IH, H-2), 8.469 (s, IH, H-8), 7.976-7.954 (IH, H-Ar, m), 7.21-7.31 (m, 3H, H-Ar), 6.50 (s, 2H, NH2); 6.054 (s IH, Η-Γ), 5.704-5.823 (2H, m OCH20), 4.760-4.819 (m, 2H, H-4', H-5'), 4.150-4.183 (m, 2H, H-3 ', H-2'), 4.010-4.049 (m, IH, H-5"), 2.75 (s, 3H), 0.986-1.100 (m, 28H).
13C NMR (400MHz, CDC13) 166.54 (C=0), 152.86 (C); 149.01 (C2), 140.5 (C6); 138.4 (C4), 132.43, 132.01, 131.95, 131.72, 130.96, 129.284 (C-Ar), 129.284 (C8), 125.671 (C5), 88.75 (OCH20), 88.54 (CI '), 81.38 (C4'), 69.059 (C2' i C3 '), 59.810 (C5'), 21.776 (CH3), 16.890-17.417 (CH3), 12.650- 13.368 (CH).
References:
1. Kierzek R. et al., Bulletin of the Polish Academy of Sciences Chemistry 35, 507-516, 1987
2. Kierzek R. et al, Nucleic Acids Symp. Ser., 18, 201-204, 1987
3. S. Czernecki, C. Georgoulis, C. Provelenghiou, Tetrahedron Lett. , 1976, 17, 3535-3536
4. E. Ohtsuka, S. Tanaka and M. Ikehara, Nucleic Acids Research Vol.1 nr 10, (1974) 5. K.K Ogilvie et.al. Proc. Natl. Acad. Sci. USA, (1988) 85, 5764-5768
6. Griffin B. E. Reese C. B., Tetrahedron Lett ., vol. 5, 2925, 1964
7. Reese C. B. et al., J. Amer. Chem. Soc, 89, 3366-3368, 1967
8. Reese C.B. et al., J. Chem. Soc. Perkin Trans. I, 2881-2885, 1988
9. Beijer B. et al., Nucleic Acids Res., 18, 2379-2390, 1990
10. Ohgi T, Masutomi Y, Ishiyama K, Kitagawa H, Shiba Y, Yano J, Org. Lett, 7, 3477-3480
(2005)
11. YoshinobuShiba,Masuda Hiroiumi, Watanabe Naoki, Ego Takeshi, TakagakiKazuchika, IshiyamaKouichi, OhgiTadaaki and Yano Junichi, Nucleic Acid Research, 2007, vol. 35, No. 10, 3287-3296
12. Lackey J.G and Damaha M.J. Nucleic Acids Symposium 52, 35-36 (2008)
Claims
1. A method for incorporating an acetal or acetal ester group into organic compounds containing an -OH group for protecting the hydroxyl function, wherein the method is based on the reaction of an organic compound containing at least one hydroxyl group, soluble in an aprotic solvent, with a compound of the general formula 1 ,
R S-CH2-0-R2 (i)
where
- Ri represents a C\. alkyl; unsubstituted or substituted benzyl or naphthyl, whereby
substituents include a C\_7 alkyl, halogen, aminoacyl;
- R.2 represents
• a Ci.i5 alkyl;
• alkyl-aryl, in which the alkyl chain contains Ci-5, whereas aryl contains from 1 to 8 unsubstituted or substituted chains, whereby substituents include a C 1.7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
• group of the general formula 2
where R3 represents:
o a C].i5 alkyl;
o ketone group;
o unsubstituted or substituted phenyl, whereby substituents include a Q.? alkyl, halogen, aminoacyl, tertiary amine group, cyano group. • group of the general formula 3
R4
Si R5
Re
(3)
wherein R4, R5 and R6 are different or the same, and represent a C1 -28 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, whereby the total number of carbon atoms in the group of the formula 3 is no less than 6 and no more than 30, in the presence of SnCU, in an aprotic solvent.
2. The method, as claimed in Claim 1, wherein the reaction is conducted in solvents selected from the group consisting of: halogen derivatives of alkanes, aromatic solvents, cyclic ethers, nitrile compounds, or a mixture of these solvents.
3. The method, as claimed in Claim 2, whereinthe reaction is conducted in solvents selected from the group consisting of: carbon tetrachloride, chloroform, dichloromethane or 1,2- dichloroethane, benzene, toluene, tetrahydrofuran, acetonitrile, or a mixture of these solvents.
4. The method, as claimed in Claim 3, wherein the reaction is conducted in 1,2-dichloroethane.
5. The method, as claimed in Claim 1 or 2 or 3 or 4, wherein SnCl4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be protected.
6. The method, as claimed in Claim 5, wherein SnCl4 is used in an amount from 1 to 6 moles per one mole of hydroxyl groups.
7. The method, as claimed in Claim 6, wherein SnCl4 is used in an amount from 2.5 to 4.5 moles per one mole of hydroxyl groups.
8. A method of protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives consisting of the incorporation of an acetal or acetal ester group, wherein it consists of the reaction between the compound of the general formula 5
where
• B represents residue of nucleobases, particularly uracil, or appropriately protected radicals of adenine, guanine, cytosine, uracil or thymine,
• Yi and Y2 are the same or different, and represent groups protecting hydroxyl functions in positions 3 ' and 5 ', in particular, they are silyl groups: triethylsilyl, tert- butyldimethyl-silyl, isopropyldimethyl-silyl, tert-butyldiphenyl-silyl, triisopropylsilyl, triphenylsilyl, methyldiisopropyl-silyl, di-tert-butylmethylsilyl
or the compound of the general formula 6
where B has the meaning defined above, and A represents group of the formulas 7, 8, 9, 10 and 1 1 :
(10) (1 1 )
compound of the general formula 1
Rl-S-CH2-0-R2 (1) where
- Ri represents a C1.6 alkyl; unsubstituted or substituted benzyl or naphthyl, whereby
substituents include a C1.7 alkyl, halogen, aminoacyl;
- R-2 represents
• a CM5 alkyl;
• alkyl-aryl, in which the alkyl chain contains C i_5, whereas aryl contains from 1 to 8 unsubstituted or substituted rings, whereby substituents include a C].7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group;
• group of the general formula 2
where R3 represents:
o a Ci_i5 alkyl;
o ketone group;
o unsubstituted or substituted phenyl, whereby substituents include a C1.7 alkyl, halogen, aminoacyl, tertiary amine group, cyano group.
• group of the general formula 3
R4
I
Si R5
R6
(3)
where R4, R5 and R6 are different or the same, and represent a Ci.28 alkyl or aryl containing from 1 to 8 rings or trimethylsilyl, whereby the total number of carbon atoms in the group of the formula 3 is no less than 6 and no more than 30, in the presence of SnCl4, in an aprotic solvent.
9. The method, as claimed in Claim 8, wherein the reaction is conducted in solvents selected from the group consisting of: halogen derivatives of alkanes, aromatic solvents, cyclic ethers, nitrile compounds or a mixture of these solvents.
10. The method, as claimed in Claim 9, wherein the reaction is conducted in solvents selected from the group consisting of: carbon tetrachloride, chloroform, dichloromethane or 1 ,2- dichloroethane, benzene, toluene, tetrahydrofuran, acetonitrile, or a mixture of these solvents.
1 1. The method, as claimed in Claim 10, wherein the reaction is conducted in 1 ,2-dichloroethane.
12. The method, as claimed in Claim 8 or 9 or 10 or 1 1 , wherein SnCl4 is used in an amount not smaller than 0.01 mole per one mole of hydroxyl groups which are intended to be substituted.
13. The method, as claimed in Claim 12, wherein SnCl4 is used in an amount from 1 to 6 moles per one mole of hydroxyl groups.
14. The method, as claimed in Claim 13, wherein SnCLt is used in an amount from 2.5 to 4.5 moles per one mole of hydroxyl groups.
15. A method for protecting the hydroxyl function, particularly in position 2' in nucleoside derivatives consisting of the incorporation of an acetal or acetal ester group, wherein the method consists of the incorporation of roups of the formulas 12 and 13 in position 2'
(12)
16. New monothioacetals of the general formula 1,
RrS-CH2-0-R2 (i)
where
Ri represents (4-chloro)phenyl,
R.2 represents o-toluoyl, benzoyl, pivaloyl.
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PL403256A PL221806B1 (en) | 2013-03-21 | 2013-03-21 | Method for introduction of acetal and acetal-ester protecting group and its use to protect a hydroxyl function |
PCT/PL2014/050012 WO2014148928A1 (en) | 2013-03-21 | 2014-03-19 | Method for incorporating protecting acetal and acetal ester groups, and its application for the protection of hydroxyl function |
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US5986084A (en) | 1997-08-18 | 1999-11-16 | Pitsch; Stefan | Ribonucleoside-derivative and method for preparing the same |
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