ZA200102683B - Crystal of diuridine tetraphosphate or salt thereof and method for preparing the same, and method for producing said compound. - Google Patents
Crystal of diuridine tetraphosphate or salt thereof and method for preparing the same, and method for producing said compound. Download PDFInfo
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- ZA200102683B ZA200102683B ZA200102683A ZA200102683A ZA200102683B ZA 200102683 B ZA200102683 B ZA 200102683B ZA 200102683 A ZA200102683 A ZA 200102683A ZA 200102683 A ZA200102683 A ZA 200102683A ZA 200102683 B ZA200102683 B ZA 200102683B
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- South Africa
- Prior art keywords
- crystals
- uridine
- salt
- tetraphosphate
- ump
- Prior art date
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- 239000013078 crystal Substances 0.000 title claims description 78
- 150000003839 salts Chemical class 0.000 title claims description 45
- 238000000034 method Methods 0.000 title claims description 31
- 150000001875 compounds Chemical class 0.000 title description 5
- 238000004519 manufacturing process Methods 0.000 title description 3
- VBGNEGYAPSWNQQ-VWDXAFRGSA-N 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione [hydroxy(phosphonooxy)phosphoryl] phosphono hydrogen phosphate Chemical compound OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1ccc(=O)[nH]c1=O.OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1ccc(=O)[nH]c1=O.OP(O)(=O)OP(O)(=O)OP(O)(=O)OP(O)(O)=O VBGNEGYAPSWNQQ-VWDXAFRGSA-N 0.000 title 1
- 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 claims description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 claims description 23
- 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 claims description 23
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 23
- 229940045145 uridine Drugs 0.000 claims description 23
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 22
- DJJCXFVJDGTHFX-ZAKLUEHWSA-N uridine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-ZAKLUEHWSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 19
- YDHWWBZFRZWVHO-UHFFFAOYSA-H [oxido-[oxido(phosphonatooxy)phosphoryl]oxyphosphoryl] phosphate Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O YDHWWBZFRZWVHO-UHFFFAOYSA-H 0.000 claims description 17
- 239000002585 base Substances 0.000 claims description 16
- BHIIGRBMZRSDRI-UHFFFAOYSA-N [chloro(phenoxy)phosphoryl]oxybenzene Chemical compound C=1C=CC=CC=1OP(=O)(Cl)OC1=CC=CC=C1 BHIIGRBMZRSDRI-UHFFFAOYSA-N 0.000 claims description 15
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 14
- 239000003513 alkali Substances 0.000 claims description 13
- PGAVKCOVUIYSFO-XVFCMESISA-N UTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-XVFCMESISA-N 0.000 claims description 11
- 229940023032 activated charcoal Drugs 0.000 claims description 11
- 150000001447 alkali salts Chemical class 0.000 claims description 11
- PGAVKCOVUIYSFO-UHFFFAOYSA-N uridine-triphosphate Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 claims description 11
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- XCCTYIAWTASOJW-UHFFFAOYSA-N UDP-Glc Natural products OC1C(O)C(COP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-UHFFFAOYSA-N 0.000 claims description 9
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 claims description 9
- 238000005571 anion exchange chromatography Methods 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 235000011180 diphosphates Nutrition 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000007858 starting material Substances 0.000 claims description 4
- ASMQGLCHMVWBQR-UHFFFAOYSA-M diphenyl phosphate Chemical compound C=1C=CC=CC=1OP(=O)([O-])OC1=CC=CC=C1 ASMQGLCHMVWBQR-UHFFFAOYSA-M 0.000 claims description 3
- 150000004677 hydrates Chemical class 0.000 claims description 2
- 239000000203 mixture Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 31
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 239000011541 reaction mixture Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 238000002441 X-ray diffraction Methods 0.000 description 12
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 12
- 150000004689 octahydrates Chemical class 0.000 description 11
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- -1 UTP from U Chemical class 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 238000000354 decomposition reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000002002 slurry Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 description 3
- 229910002483 Cu Ka Inorganic materials 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 229920001429 chelating resin Polymers 0.000 description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical class OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 3
- 150000002576 ketones Chemical class 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 229940048084 pyrophosphate Drugs 0.000 description 3
- 150000004685 tetrahydrates Chemical class 0.000 description 3
- CYSGHNMQYZDMIA-UHFFFAOYSA-N 1,3-Dimethyl-2-imidazolidinon Chemical compound CN1CCN(C)C1=O CYSGHNMQYZDMIA-UHFFFAOYSA-N 0.000 description 2
- JYYNAJVZFGKDEQ-UHFFFAOYSA-N 2,4-Dimethylpyridine Chemical compound CC1=CC=NC(C)=C1 JYYNAJVZFGKDEQ-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 229920000388 Polyphosphate Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 150000004292 cyclic ethers Chemical class 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003172 expectorant agent Substances 0.000 description 2
- 230000003419 expectorant effect Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 239000001205 polyphosphate Substances 0.000 description 2
- 235000011176 polyphosphates Nutrition 0.000 description 2
- 239000012048 reactive intermediate Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- BMVXCPBXGZKUPN-UHFFFAOYSA-N 1-hexanamine Chemical class CCCCCCN BMVXCPBXGZKUPN-UHFFFAOYSA-N 0.000 description 1
- XWKFPIODWVPXLX-UHFFFAOYSA-N 2-methyl-5-methylpyridine Natural products CC1=CC=C(C)N=C1 XWKFPIODWVPXLX-UHFFFAOYSA-N 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 229920001890 Novodur Polymers 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- SBVYRQFJZOKNGO-XVFCMESISA-N [[(2r,3s,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [hydroxy(phosphonooxy)phosphoryl] hydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 SBVYRQFJZOKNGO-XVFCMESISA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000011260 aqueous acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- JQVDAXLFBXTEQA-UHFFFAOYSA-N dibutylamine Chemical class CCCCNCCCC JQVDAXLFBXTEQA-UHFFFAOYSA-N 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- WJFREWBMJPXLBS-UHFFFAOYSA-N n,n-dibutylbutan-1-amine;phosphono dihydrogen phosphate Chemical compound OP(O)(=O)OP(O)(O)=O.CCCCN(CCCC)CCCC WJFREWBMJPXLBS-UHFFFAOYSA-N 0.000 description 1
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Description
" h ~ 20012683
The present invention relates to stable crystals of
P!,P*-di(uridine 5'-)tetraphosphate (U,Py) or a salt thereof which are useful as an active ingredient of an expectorant or a therapeutic agent for pneumonia; a process for producing . the crystals; and a process for efficiently producing U,P, or a salt thereof.
A tetrasodium salt of P!,P*-di(uridine 5'- : )tetraphosphate (U;P,*4Na) represented by the following formula (I): 0 0
HN 1] 0 0 0 iN 8 on : 1) 0A N HOF ~0 =F -0—F —0~F —0-Ci, 0 NN gg | in one ONa ONa < J
No bb
OH OH (1) : has an expectoration-inducing action and is a compound which is expected to be developed as an expectorant or a therapeutic agent for pneumonia (e.g., U.S. Patent Nos. 5,789,391, 5,763,447, and 5,635,160).
N ha Until now, U,P, has not been obtained in crystal form, and has been prepared only in the form of a lyophilized : product (see Example 1 of WO 99/05155). Typical U.P, produced by conventional method has a purity as low as 90%, and contains by-products. Examples of by-products include nucleoside 5'-(poly)phosphates such as uridine 5'- tetraphosphate (UP,), uridine 5' -triphosphate (UTP), uridine 5'-diphosphate (UDP), and uridine 5'-monophosphate (UMP); and dinucleoside polyphosphates such as P!,P*-di(uridine 5°'- )triphosphate (U,P;) and P!,P*-di (uridine 5'-)diphosphate (UzP;). Particularly, it is difficult to separate nucleoside 5'-(poly)phosphates such as UTP from U,Ps, arid highly purified U.P, has been produced only with great difficulty through a conventional purification method: i.e., ion- exchange chromatography (WO 99/05155, Biochimica er
Biophysica Acta, 438, (1976) 304-309).
The above purified and lyophilized product has disadvantages such as high hygroscopicity. Therefore, preparation of a pharmaceutical from U.P, must be carried out in a special apparatus in which moisture is well controlled.
Even after preparation of a pharmaceutical, the product must be wrapped tightly. In addition, since the pharmaceutical has a very short available period due to poor stability of the lyophilized preparation, obtaining highly purified and stable U,P; crystals has been desired.
U,Py is synthesized from uridine 5'-monophosphate (UMP) by use of an activating agent such as diphenyl
Lo 20012683 bo. phosphochloridate (DPC) and a phosphorylating agent such as a pyrophosphate (PPi). However, a conventional process provides a low synthesis yield; i.e., as low as approximately wt.% (Example 4B of WO 99/05155), and can never serve as a practical process. Accordingly, development of a process for producing U,P; at high yield and on a large scale has also been desired.
In view of the foregoing, an object of the present invention is to provide stable crystals of U,P, or a salt thereof. Another object of the invention is to provide a process for producing the crystals. Still another object of the invention is to provide a process for efficiently producing U,P, on a large scale. -
The present inventors have conducted earnest studies on a method for purifying U,P, and a process for synthesizing
U,P4 from UMP. The inventors have found that U.P, purified through anion exchange chromatography and chromatography using activated charcoal (activated-charcoal chromatography) : can be easily crystallized and that use of specific reaction conditions has the effect of drastically increasing the yield of U,P4 in the synthesis of UzPs or a salt thereof from UMP serving as a starting material and by use of DPC and PPi.
The present invention has been achieved on the basis of these findings.
Accordingly, the present invention provides crystals of
“ P',P*-di (uridine 5'-)tetraphosphate or a salt thereof.
The present invention also provides crystals of Pp!,pi- di(uridine 5'-)tetraphosphate tetrasodium salt having a crystal structure showing characteristic peaks in X-ray diffraction employing Cu-Ka rays in the vicinity of the diffraction angles (20) of 5.9, 11.5, 12.4, 15.4, 17.2, 18.0, 19.8, and 20.5(°).
The present invention also provides crystals of p!,pt- di (uridine 5'-)tetraphosphate tetrasodium salt having a crystal structure showing characteristic peaks in an IR absorption spectrum at wavelengths in the vicinity of 1690, 1277, 1233, 1146, 1116, and 890 (cm). :
The present invention also provides a process for producing crystals of P',P*-di (uridine 5'-)tetraphosphate or a salt thereof, which process comprises purifying crude
P',P*-di(uridine 5'-)tetraphosphate or a salt thereof through anion exchange chromatography and activated-charcoal chromatography and adding a hydrophilic organic solvent to a solution of purified P',P*-di(uridine 5'-)tetraphosphate or a salt thereof, to thereby precipitate crystals.
The present invention also provides a process for producing P',P*-di(uridine 5'-)tetraphosphate or a salt thereof from uridine 5'-monophosphate (UMP) serving as a starting material and by use of diphenyl phosphochloridate (DPC) and a pyrophosphate (PPi), which process comprises at least one cof the following treatment steps: (a) adding UMP diphenylphosphate (UMP-DPP) in divided
* portions during a step of reaction of UMP-DPP with a PPi- organic alkali salt; (b) carrying out a step of reaction of UMP~-DPP with a
PPi-organic alkali salt in the presence of a base; and (c) further treating the synthesized U,Py, with an alkali.
Fig. 1 shows an X-ray diffraction spectrum of crystalline. U,P,*4Na tetrahydrate crystallized from ethanol solution.
Fig. 2 shows an X-ray diffraction spectrum of crystalline U,P,'4Na octahydrate crystallized from ethanol solution. :
Fig. 3 shows an X-ray diffraction spectrum of U,P, obtained through lyophilization.
Fig. 4 is.a photograph showing crystal form of crystalline U,P,'4Na octahydrate crystallized from ethanol solution. The photograph was taken under a polarizing microscope (magnification: 440), wherein 1 cm in the image corresponds to 23 um.
Fig. 5 shows an IR absorption spectrum of crystalline
UzP4 4Na octahydrate crystallized from ethanol solution.
Fig. 6 shows an IR absorption spectrum of U,P4 obtained through lyophilization.
Fig. 7 shows an X-ray diffraction spectrum of crystalline U;P;'4Na octahydrate crystallized from methanol solution.
" Best Mode for Carrying Out the Invention
The crystals of U,P; or a salt thereof according to the present invention are obtained through purification of crude
U;P4 or a salt thereof by use of specific means, and addition of a hydrophilic organic solvent to a solution of purified
UP; or a salt thereof, to thereby precipitate the solute as crystals. The present invention will next be described in terms of (1) purification of U,P, or a salt thereof and (2) crystallization of U,P; or a salt thereof. (1) Purification of UP; or a salt thereof
U,Py, or a salt thereof can be purified through anion exchange chromatography and activated-charcoal chromatography performed in combination. Although the two chromatography techniques may be performed in arbitrary sequence, anion exchange chromatography preferably precedes activated- charcoal chromatography, in view of improvement of the purity of U,Py.
A styrenic or acrylic resin may be used as an anion- exchanging resin in the above-described chromatography techniques. Examples of resins which may be used include strongly basic anion-exchanging resins such as AMBERLITE IRA 402 (Rohm & Haas Co.), DIAION PA-312, and DIAION SA-11A (Mitsubishi Chemical Co. Ltd.), and weakly basic anion- exchanging resins such as AMBERLITE IRA 67 (Rohm & Haas Co.) and DIAION WA-30 (Mitsubishi Chemical Co. Ltd. ).
The activated charcoal may be in the form of chromatography-grade activated charcoal which is crushed or x shaped into particles, and may include commercially available products (e.g., those of Wako Pure Chemical Industries, Ltd. and Futamura Kagaku Kogyo).
Chromatography may be carried out in a batch manner, Or by use of a column. When the column chromatography is carried out, an aqueous acid solution or a mixture thereof with a salt having enhanced ionic strength, such as sodium chloride, may be used as an eluent for anion exchange chromatography; and water or an aqueous solution of alkali such as sodium hydroxide may be used as an eluent for activated-charcoal column chromatography. A small-scale preliminary test may be conducted in order to appropriately determine the concentration of each eluent from the range of 0.001 M to 10 M. (2) Crystallization of U,P, or a salt thereof
UP; or a salt thereof is crystallized through addition of an hydrophilic organic solvent to a solution containing the thus-purified U,P, or a salt thereof.
Examples of the hydrophilic organic solvents which may be used include alcohols having six or fewer carbon atoms, such as methanol and ethanol; ketones such as acetone; ethers such as dioxane; nitriles such as acetonitrile: and amides such as dimethylformamide. Of these, alcohols, especially ethanol, are particularly preferred.
More specifically, a solution of the thus-purified UP, or a salt thereof, or a slurry obtained through concentration of the solution, is optionally treated to thereby adjust the
* PH to 6-9, and a hydrophilic organic solvent is added to the solution or slurry at 60°C or less to thereby precipitate the . solute as stable U,P, Crystals.
The thus-obtained U,P, crystals of the present invention contain (1) UyP, in an amount of 95% or more and (2) other homologous compounds in an amount. of 5% or less.
In the present invention, other homologous compounds include nucleoside 5'-{poly)phosphates such as UP,, UTP, UDP, and UMP; and dinucleoside polyphosphates such as U,P; and U,P,.
More preferably, U.P4 crystals contain (1) UP; in an amount of 97% or more, (2) UDP in an amount of 1% or less, and (3) UTP in an amount of 1% or less. Particularly preferably, U,P; crystals contain (1) UyP4 in -an amount of 98% or more, (2) UDP in an amount of 0.5% or less, and (3) UTP in an amount of 0.5% or less.
Such highly purified U,P, crystals may be in the form of a salt, hydrate, or hydrate salt. Examples of the salts include pharmaceutically acceptable salts such as alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and ammonium salts. The U,P;, may be substituted with 1-4 metal atoms to form a salt.
The above hydrate may comprise 3-8 molecules of water which are bound to or adhere to one molecule of U,Ps, and the above hydrate salt may comprise 3-8 molecules of water which are bound to or adhere to one molecule of an alkali metal salt of U,P,.
= Examples of preferred U.Py crystals include U,P,:4Na crystals and hydrates thereof. The U,P,*4Na crystals show characteristic peaks in X-ray diffraction employing Cu-Ka rays in the vicinity of the diffraction angles (20) of 5.9, 11.5, 12.4, 15.4, 17.2, 18.0, 19.8, and 20.5(°) (error range %0.1°), and show characteristic peaks in an IR absorption spectrum at wavelengths in the vicinity of 1690, 1277, 1233, 1146, 1116, and 890 (cm™*) (error range +2 cm!). Furthermore, the U,P,*4Na crystals are easy to handle and remarkably useful as compared with conventicnal lyophilized products, since the crystals are stable under high temperature, high humidity conditions and the water content of‘ the crystals is stabilized at 5-15 wt.%, to thereby suppress -further hygroscopicity.
As described above, the thus-obtained U,P4'4Na crystals of the present invention contain (1) U,Ps, in an amount of 95% or more and (2) other homologous compounds in an amount of 5% or less. In addition, as described above, examples of the preferred U,P, crystals include such crystals containing (1)
U;Py in an amount of 97% or more, (2) UDP in an amount of 1% or less, and (3) UTP in an amount of 1% or less, particularly crystals containing (1) U,Ps in an amount of 98% or more, (2)
UDP in an amount of 0.5% or less, and (3) UTP in an amount of 0.5% or less.
Furthermore, the crystals of U,P, or a salt thereof according to the present invention also include tautomers thereof. i
S h The crystals of U.P, or a salt thereof according to the present invention are optionally dried through a conventional method such as drying under reduced pressure, drying under air-flow, or drying by heating, and are subsequently placed in a container (e.g., bottle, pouch, can, ampoule). Packing in the container may be carried out such that the container is open, closed, airtight, or sealed. The open condition is not preferred, in view of maintenance of storage stability of the crystals.
Next, an efficient process for synthesizing U,P, will be described.
Conventionally, U,P, or a salt thereof Has predominantly been synthesized from uridine 5'-monophosphate (UMP) serving ds a starting material and by use of an activating agent such as diphenyl phosphochloridate (DPC) and a phosphorylating agent such as & pyrophosphate (PPi). Specifically, DPC and tributylamine are added to a tributylamine salt of UMP, to thereby produce UMP diphenylphosphate (UMP-DPP) serving as a reactive intermediate, which is reacted with tributylamine pyrophosphate (TBA-PPi), to thereby obtain U,P, or a salt . thereof at a yield of approximately 9.6% (Example 4B of WO 99/05155).
The process according to the present invention is characterized in that at least one of the following treatment steps is carried out: (a) adding UMP-DPP in divided portions during a conventional step of reaction of UMP-DPP with a PPi- organic alkali salt; (b) carrying out reaction of UMP-DPP
” + with a PPi-organic alkali salt in the presence of a base; and {c) further treating the synthesized U,P4 with an alkali.
Two or more of the above treatment steps may be combined.
The step "(a) adding UMP-DPP in divided portions™ refers to addition of UMP-DPP, which must be provided in an amount by mol of at least twice that of a PPi-organic alkali salt, in several portions rather than in a single portion.
For example, a PPi-organic alkali salt is reacted with an equimol amount of UMP-DPP and the step is repeated. Although no particular limitation is imposed on the number of portions of UMP-DPP, 2-3 portions are preferred in view of increase of the yield. i.
Examples of the PPi-organic alkali salts include a hexylamine salt, a dibutylamine salt, a triethylamine salt, and a tributylamine salt. In reaction with UMP-DPP, the
PPi~organic alkali salt may be dissolved in a solvent.
Examples of the solvents include amides such as DMF, DMAC, and formamide; cyclic ethers such as dioxane and tetrahydrofuran; ketones such as acetone; and . dimethylimidazolidinone, hexamethylphosphoric triamide, dimethylsulfoxide, acetonitrile, or a mixture of two or more of these. Subsequently, UMP-DPP is added to the solution, and the mixture is allowed to react at room temperature for approximately 30 minutes to five hours.
The step "(b) carrying out reaction of UMP-DPP with a
PPi-organic alkali salt in the presence of a base" refers to a reaction carried out in the presence of a base. Examples of the bases include pyridine bases such as pyridine, 2,6- lutidine, 2,4-lutidine, o-,B-,y-picoline, 2,4- : dimethylaminopyridine, and oa-,f-,y-collidine, with pyridine being particularly preferred. A basic solvent for the reaction is also included in the bases used in the present invention. The concentration of the base is nct particularly limited. The base is preferably added in an amount of 6 eguivalents or more based on UMP, more preferably 18 equivalents. or more.
Furthermore, the step "(c) further treating the synthesized U,;P, with an alkali" refers to quenching of a liguid containing synthesized U,P; with water and treating the mixture with a solution of an organic or-inorganic alkali such as sodium hydroxide, ammonia, potassium hydroxide, pyridine, triethyalmine, or sodium carbonate. Conventionally, the quenched liquid as such is purified directly. However, the treatment with an alkali enables improvement of the isolation yield of U,P; as compared with the conventional method. : . i
In the treatment with an alkali, a liquid containing i synthesized U,P; is guenched with water, and an alkali is added to the mixture such that the pH of the mixture becomes approximately 8-13, preferably 10-12. The mixture is allowed to react at room temperature for approximately 10 minutes to five hours.
UMP-DPP can be synthesized from UMP through a conventional method. For example, a UMP trialkylamine salt ’ 12
AMENDED SHEET 26.10.2001
<4 : such as a UMP tributylamine salt prepared through a conventional method is dissolved in a solvent. Examples of : the solvents include amides such as DMF and dimethylacetamide (DMAC): cyclic ethers such as dioxane and tetrahydrofuran; ketones such as acetone; and dimethylimidazolidinone, hexamethylphosphoric triamide, or a mixture thereof.
Subsequently, DPC and, if needed, a trialkylamine are added to the solution, and the mixture is allowed to react at room temperature. for approximately 30 minutes to five hours, to thereby synthesize UMP-DPP serving as a reactive intermediate.
The present invention will next be described in more detail by way of examples.
Example 1 Synthesis of U,P; or a salt thereof (1) Effect of .a base
DMAC (8 mL) was added to a dehydrated uridine 5°- monophosphate tributylamine salt (UMP-TBA) (6.2 mmol), and
DPC (1.7 mL) was added dropwise to the mixture with stirring.
The thus-obtained mixture was reacted at room temperature for one hour to thereby form UMP-DPP, after which TBA (7.6 mL) was added to the reaction mixture, which was stirred for another 10 minutes. Meanwhile, dehydrated TEA-PPi (3.0 mmol) was dissolved in pyridine (1.7 mL), and the thus-prepared solution was added to the UMP-DPP reaction mixture.
Subsequently, the mixture was stirred at room temperature for three hours, and water was added to the mixture to thereby
2 stop the reaction. The obtained reaction mixture was analyzed by HPLC (at 262 nm), which showed that the target : U.P, was obtained at a yield of 18.3%.
As is apparent from the result, when UMP-DPP is reacted with TEA-PPi in the presence of a base (pyridine), U,P, may be synthesized at about twice the yield obtained through a conventional method. (2) Effect of combination use of base and alkali treatment
DMAC (8 mL) was added to a dehydrated uridine 5°- monophosphate tributylamine salt (UMP-TBA) (6.2 mmol), and
DPC (1.7 mL) was added dropwise to the mixture with stirring.
The thus-obtained mixture was reacted at room temperature for one hour toc thereby form UMP-DPP, after which, TBA (7.6 mL) was added to the reaction mixture and the mixture was stirred ’ for another 10 minutes. Meanwhile, dehydrated TEA-PPi (3.0 mmol) was dissolved in pyridine (1.7 mL), and the thus- prepared solution was added to the UMP-DPP reaction mixture.
Subsequently, the mixture was stirred at room temperature for three hours, and water was added to the mixture to thereby stop the reaction. A 30% sodium hydroxide solution was added to the above-obtained reaction mixture so as to adjust pH to 11.0, and the mixture was allowed to stand overnight. The obtained reaction mixture was analyzed by HPLC (at 262 nm), which showed that the target U,P4; was obtained at a yield of 26" 75.
As 1s apparent from the result, when UMP-DPP is reacted with TEA-PPi in the presence of a base (pyridine) and further
RE alkali treatment is performed, U.P; may be synthesized at about three times the yield obtained through a conventional : method. (3) Effect of base and addition of UMP-DPP in several portions
Formamide (1.5 mL) and pyridine (1.5 mL) were added to a triethylamine salt of dehydrated pyrophosphoric acid (TEA-
PPi)} (6 mmol), and the mixture was stirred in a vessel.
Meanwhile, in another vessel, DMAC (4.3 mL), dioxane (4.8 mL), and tributylamine (TBA) (5.8 mL) were added to a dehydrated uridine 5’ -monophosphate tributylamine salt (UMP-TBA) (12 mmol), and the mixture was stirred. Subsequently, DPC (2.5 mL) was added dropwise to the mixture, and the thus-obtained mixture was further stirred at room temperature for one hour, to thereby form UMP-DPP. Half of the UMP-DPP reaction mixture was added dropwise to the vessel containing TEA-PPi, and reaction was allowed to proceed at room temperature for one hour. Subsequently, pyridine (1.5 mL) was added to the mixture, and the remaining UMP-DPP reaction mixture was added dropwise to the vessel. The thus-obtained reaction mixture was further reacted at room temperature for one hour, and water was added to the mixture to thereby stop the reaction.
The obtained reaction mixture was analyzed by HPLC (at 262 nm), which showed that the target U,P, was obtained at a vield of 29.5%.
As is apparent from the result, when UMP-DPP is added in two portions and UMP-DPP is reacted with TEA-PPi in the
A presence of a base (pyridine), U.P; may be produced at about three times the yield obtained through a conventional method.
As is also apparent from the result, addition of UMP-DPP in two portions provides a yield of 29.5%, which is about 1.6 times the yield obtained in (1) above (18.3%). (4) Effect of alkali treatment
Formamide (1.5 mL) and pyridine (1.5 mL) were added to a triethylamine salt of dehydrated pyrophosphoric acid (TEA-
PPi) (6 mmol), and the mixture was stirred in a vessel.
Meanwhile, in another vessel, DMAC (4.3 mL), dioxane (4.8 mL), and tributylamine (TBA) (5.8 mL) were added to a dehydrated uridine 5°'-monophosphate tributylamine salt {UMP-TBA) (12 mmol), and the mixture was stirred. Subsequently, DPC (2.5 mL) was added dropwise to the mixture, and the thus-obtained mixture was further stirred at room temperature for one hour, to thereby form UMP-DPP. Half of the UMP-DPP reaction mixture was added dropwise to the vessel containing TEA-PPi, and reaction was allowed to proceed at room temperature for one hour. Subsequently, pyridine (1.5 mL) was added to the mixture, and the remaining UMP-DPP reaction mixture was added dropwise to the vessel. The thus-obtained reaction mixture was further reacted at room temperature for one hour, and water was added to the mixture to thereby stop the reaction.
A 30% Sodium hydroxide solution was added to the above- obtained reaction mixture so as to adjust pH to 11.0, and the mixture was allowed to stand overnight. The obtained reaction mixture was analyzed by HPLC (at 262 nm), which
* showed that the target U.P; was obtained at a yield of 32.2%.
As is apparent from the result, when alkali treatment . is added to the above-described (3). the yield is increased by about 10%; i.e., from 29.5% to 32.2%.
Example 2 Production of U,P.*4Na crystals
Formamide (10 mL) and pyridine (15 mL) were added to a triethylamine salt of dehydrated pyrophosphoric acid (TEA-
PPi) (40.5 mmol), and the mixture was stirred in a vessel.
Meanwhile, in another vessel, DMAC (50 mL), dioxane (34 mL), and tributylamine (TBA) (30 mL) were added to a dehydrated uridine 5°‘-monophosphate tributylamine salt (UMP-TBA) (80 mmol), and the mixture was stirred. Subsequently, DPC (17.8 mL) was added dropwise to the mixture, and the thus-obtained mixture was further stirred at room temperature for one hour, to thereby form UMP-DPP. Half of the UMP-DPP reaction mixture was added dropwise to the vessel containing TEA-PPi, and reaction was allowed to proceed at room temperature for one hour. Subsequently, 4-dimethylaminopyridine (DMAP) (50 mg) and pyridine (15 mL) were added to the mixture, and the remaining UMP-DPP reaction mixture was added dropwise to the vessel. The thus-obtained reaction mixture was further reacted at room temperature for two hours, and water was added to the mixture to thereby stop the reaction. The reaction mixture was diluted with water to a total volume of 700 mL, and a sodium hydroxide solution was added to the solution to thereby adjust pH to 10. The solution was concentrated to 200 mL, and ethanol (250 mL) was added to the
4 concentrated solution with stirring. The solution was allowed to stand at 4°C overnight, and the supernatant was . removed by decantation. The thus-obtained solution was diluted with water to a total volume of 250 mL, and was analyzed by HPLC (at 262 nm), which showed that the target
U;P4 was obtained at a yield of 30.0%.
The above-obtained solution (110 mL) was diluted with water to a total volume of 2000 mL and the diluted solution was applied to a weak anion exchange column (AMBERLITE IRA- 67) (Cl type) (200 mL). Subsequently, the column was washed with water, and by-products were eluted with 0.18 M hydrochloric acid, after which the target U,P, was eluted with a 0.005 M hydrochloric acid solution containing 0.35 M
NaCl (recovery percentage: 82.7%).
A sodium hydroxide solution was added to the thus- obtained eluate to thereby adjust pH to 2.5. . Subsequently, the eluate was applied to an activated charcoal column (Taiko
SGP), and the column was washed with water and eluted with a 0.05 M sodium hydroxide solution (recovery percentage: 84.9%).
The pH of the thus-obtained eluate was adjusted to 7.6, and the eluate was concentrated to 38 ml. Subsequently, ethanol (57 mL) was added to the concentrated solution to thereby obtain 3.1 g of U;P,*4Na crystals (water content: 7.8%) (isolation yield: 18.4%). (Physical properties of U,P,‘4Na crystals)
The UzP4-4Na crystals prepared in Example 2 were dried at 60°C for four hours by use of a forced-air drier, and
! subjected to instrumental analysis. In addition, a lyophilized product of U,P,:4Na was prepared in the same way : as in the method described in Example 1 of WO 99/05155, and the thus-prepared product was compared with the crystals in terms of physical properties. (1) Instrumental analysis 1) Analysis of purity
The U,P4-4Na crystals obtained in Example 2, and fractions containing U,P, after purification through each chromatography were subjected to analysis of purity by means of high performance liquid chromatography. The results are : shown in Table 1. Conditions for the high performance liquid chromatography are described below. .
Column: HITACHIGEL #3013-N (product of Hitachi
Keisokuki Service)
Eluent: 10% CH3CN, 0.18 M NH,Cl, 0.03 M KH,PO;, and 0.03
M K,HPO,
Detection method: UV detection at 262 nm
Table 1 :
Proportions of substances (wt.$%)
UzP4 and Reac- After anion After activated After its ana- | tion exchange crystal- , charcoal column . . logues mixture | column lization
UMP 7.5 0.5 0.3 (-)
U.P 29.8 0.4 (=) (-) ubDPp 2.5 0.1 0.2 0.1
U,P3 17.6 0.2 (-) (-)
UTP 13.0 0.8 0.4 0.1
U.P, 21.0 97.9 98.6 99.8
UP, 7.3 (-) (-) (-) (-): Below detectable limit
2) Water content : The U,P,*4Na crystals were subjected to measurement of water content by means of the Karl Fischer method, to thereby show a water content of 5-15 wt.%, which varied in accordance with the degree of drying. The results apparently show that three to eight water molecules bind or adhere to one U.P, molecule. 3) Melting point
The U,P,* 4Na crystals were subjected to measurement of melting point by means of a conventional method, to thereby provide a decomposition point of about 223°C: The decomposition point of the lyophilized product was about 220°C, 4) X-ray diffraction
The U,P,*4Na crystals were subjected to measurement of
X-ray diffraction by use of an X-ray diffraction apparatus (Model: RINT2500V, product of Rigaku Denki) under the following conditions. The thus-obtained X-ray diffraction spectra are shown in Figs. 1 and 2, and the peak data are shown in Tables 2 and 3. (Conditions for measurement)
X-ray tube: Cu-Ka
X-ray output: 50 kV-300 mA
Scanning rate: 4.0°/minute
Scanning interval: 0.02°
Angle measuring range: 2-40°
TE
Slit: DS-0.5°, RS-0.15 mm, SS-0.5°
Pre-treatment: Grinding by use of an agate mortar : Table 2
Peak 26 Relative
No. °) intensity (I/I,) 1 5.96 100 11.58 38 6 12.42 79 15.42 48 13 17.18 45 18.04 55 16 19.86 84 17 20.56 73 18 21.18 51 19 21.40 51 25.22 42 29 27.52 45 27.98 47 : 30.60 40
Table 3
Peak 20 Relative
No. ) intensity (1/710) 1 5.96 1c0 5 11.56 41 6 12.42 90 10 15.42 51 12 17.18 48 : 14 18.04 63 15 19.86 90 16 20.58 90 17 21.20 56 18 21.42 59 24 25.20 50 29 27.54 56 30 27.96 57 35 30.60 ' 48
Fig. 1 and Table 2 show the data for the crystals of
U.P, 4Na tetrahydrate, and Fig. 2 and Table 3 show the data
+ for the crystals of U2P4°4Na octahydrate. In addition, the
X-ray diffraction spectrum of the lyophilized product is : shown in Fig. 3 as a reference. 5) Hygroscopicity
UP, 4Na crystals (octahydrate) having a water content of approximately 14% were allowed to stand for nine days under the following conditions: a) 25°C and a relative ~ humidity of 57%; b) 25°C and a relative humidity of 75%; and
Cc) 40°C and a relative humidity of 75%. No decomposition or change in weight was observed in the above three cases. The crystals have proven to be stable and to have no hygroscopicity. U,P,*4Na crystals (tetrahydrate) having a water content of approximately 8% were allowed to stand for nine days under conditions of 40°C and a relative humidity of 75%. In this case, the water content increased to 14-15%.
However, the water content did not increase further, and the crystals were stabilized.
In contrast, when a lyophilized product (initial water content: approximately 1 %) was allowed to stand for nine days under conditions of 40°C and a relative humidity of 75%, the water content increased gradually, and on the seventh day of storage the product assumed a mud-like state due to deliquescence. 6) Stability
UP4*4Na crystals (octahydrate) and a lyophilized product were placed in respective bottles, which were then sealed and allowed to stand for 13 days at 60°C (acceleration
A test). No decomposition of the crystals was observed, while partial decomposition of the lyophilized product was confirmed through observation of a purity lowering of U,P,+4Na of approximately 1.4%. 7) Crystal form
Fig. 4 shows a photograph of a typical crystal form of
U.P, 4Na (octahydrate) crystals. g) IR absorption spectra
IR spectra of UzP4-4Na crystals (octahydrate) and a lyophilized product were measured in a customary manner by use of a JASCO 5000 Spectrophotometer. The results are shown in Figs. 5 and 6. The lyophilized product of U.P; 4Na exhibits peaks at 3416, 1702, 1266, 1116, 1079, and 906 (cm 'y (Fig. 6), whereas U,P4- 4Na crystals exhibit peaks at 3386, 1690, 1277, 1233, -1146, 1116, and 890 (cm™) (Fig. 5).
Example 3 Production of U,P,-4Na octahydrate crystals
The fraction containing UzP, obtained through treatment with a column in Example 2 was concentrated to thereby prepare a slurry, and pH thereof was adjusted to 7.0.
Methanol was gradually added to the slurry with stirring, and the slurry was further stirred with cooling to 10°C, to thereby precipitate U,P.4Na crystals.
The water content of the thus-obtained and dried
U,Ps-4Na crystals was measured through the Karl Fischer method, to thereby determine that the crystals are octahydrate. The X-ray diffraction spectrum of the crystals is shown in Fig. 7, and peak data thereof are shown in Table . 23 .
AMENDED SHEET 26.10.2001
¥
Table 4 .
Relati : Peak 26 . ve
No (°) intensity ’ (1/1) 1 5.96 100 11.58 30 6 12.42 77 8 15.40 50 } 17.20 38 . 11 18.04 38 12 19.84 82 . 13 20.62 66 } 14 21.42 48 19 © 25.38 36 23 27.58 39 24 27.98 37 30.64 33
As described hereinabove, the crystals of U;P4 or a salt thereof obtained through the process according to the present invention have high purity and stability and less hygroscopicity as compared with a lyophilized product, to thereby serve as a useful raw material for preparing a pharmaceutical.
The process for producing UP, or a salt therect : according to the present invention realizes high yield and enables large-scale synthesis. ’ 24
AMENDED SHEET 26.10.2001
Claims (1)
- ‘e Claims1. Crystals of Pl, P’-di (uridine 5'-) tetraphosphate or a salt therof.2. Crystals as described in claim 1, which have a purity of at least 95%.3. Crystals as described in claim 1, which have a purity of at least 97% and which contain uridine 5'- triphosphate and uridine 5'-diphosphate each in an amount of 1% or less. 4, A process for producing crystals of P!,P*-di (uridine 5'-)tetraphosphate or a salt thereof, which process comprises purifying crude P!,P*-di (uridine 5'-)tetraphosphate or a salt thereof through anion exchange chromatography and activated- charcoal chromatography and adding a hydrophilic organic solvent to a solution of purified P', P*-di (uridine 5'-) tetraphosphate or salt thereof, to thereby precipitate the solute as crystals.5. A process for producing p!, P~di (uridine 5'-) tetraphosphate or a salt thereof according to claim 4, wherein purification comprises anion exchange chromatography follwed by activated-charcoal chromatography.6. A process for producing pl, P*-di (uridine 5'-) tetraphosphate or a salt thereof from uridine 5'- monophosphate (UMP) serving as a starting material and by use of diphenyl phosphochloridate (DPC) and a pyrophosphate (PPi), which process comprises at least one of the following treatment steps (a) to (cc): (a) adding UMP diphenylphosphate (UMP-DPP) in divided portions during a step of reaction of UMP-DPP with a PPi- organic alkali salt; AMENDED SHEET 26.10.2001Y (b} carrying out a step of reaction of UMP-DPP with a PPi-organic alkali salt in the presence of a base; and (c) further treating the synthesized U,P, with an alkali.7. A process for producing P',P‘-di (uridine 5'-) tetraphosphate or a salt thereof according to claim 6, comprising steps (a) to (c) as defined in claim 6.8. Crystals of PY, P-di (uridine 5'-)tetraphosphate:4Na.9g. Crystals of P',P*-di (uridine 5'-)tetraphcsphate-4 Na hydrates.10. Crystals as described in claim 9, comprising 3-8 molecules of water which are bound to or adhere to one molecule of P!,P%-di (uridine 5'-)tetraphosphate-4 Na.11. Crystals as described in claim 9, comprising 4 molecules of water which are bound to or adhere to one molecule of P',P!-di (uridine 5'-)tetraphosphate-4 Na.12. Crystals are described in claim 9, comprising 8 molecules of water which are bound to or adhere to one molecule of P!,P*-di (uridine 5'-)tetraphosphate-4 Na.13. Crystals as described in any one of claims 8-12, which have a purity of at least 95%.14. Crystals as described in any one of claims 8-12, which have a purity of at least 97% and which contain uridine 5'-triphosphate and uridine 5'-diphosphate each in an amount of 1% or less.15. Crystals according to claim 1, substantially as herein described with reference to Example 2.16. A process according to claim 4, substantially as herein described with reference to Example 2.17. A process according to claim 6, substantially as herein described with reference to Example 1. 26 AMENDED SHEET 26.10.2001 v18. Crystals according to claim 8, substantially as herein described with reference to Example 2.19. Crystals according to claim 9, substantially as herein described with reference to Example 2. 27 AMENDED SHEET 26.10.2001
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| US5789391A (en) * | 1996-07-03 | 1998-08-04 | Inspire Pharmaceuticals, Inc. | Method of treating sinusitis with uridine triphosphates and related compounds |
| US5763447C1 (en) * | 1996-07-23 | 2002-05-07 | Inspire Pharmaceuticals | Method of preventing or treating pneumonia in immobilized patients with uridine triphosphates and related compounds |
| JP3723227B2 (en) * | 1997-07-25 | 2005-12-07 | インスパイアー ファーマシューティカルズ，インコーポレイティド | Process for large-scale production of di (uridine 5 ')-tetraphosphate and its salts |
-
1999
- 1999-10-01 CN CNB2005100228292A patent/CN100354296C/en not_active Expired - Lifetime
-
2001
- 2001-04-02 ZA ZA200102683A patent/ZA200102683B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN100354296C (en) | 2007-12-12 |
| CN1781928A (en) | 2006-06-07 |
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