CN1613864A - Method for purifying and preparing nucleoside triphosphate derivative - Google Patents
Method for purifying and preparing nucleoside triphosphate derivative Download PDFInfo
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- CN1613864A CN1613864A CN 200310108337 CN200310108337A CN1613864A CN 1613864 A CN1613864 A CN 1613864A CN 200310108337 CN200310108337 CN 200310108337 CN 200310108337 A CN200310108337 A CN 200310108337A CN 1613864 A CN1613864 A CN 1613864A
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- nucleoside
- exchange resin
- nucleoside triphosphate
- derivative
- bicarbonate
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- 239000002777 nucleoside Substances 0.000 title claims abstract description 77
- -1 nucleoside triphosphate derivative Chemical class 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims description 17
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 21
- 239000001226 triphosphate Substances 0.000 claims abstract description 11
- 235000011178 triphosphate Nutrition 0.000 claims abstract description 11
- 239000011347 resin Substances 0.000 claims abstract description 10
- 229920005989 resin Polymers 0.000 claims abstract description 10
- WVPKAWVFTPWPDB-UHFFFAOYSA-N dichlorophosphinic acid Chemical class OP(Cl)(Cl)=O WVPKAWVFTPWPDB-UHFFFAOYSA-N 0.000 claims abstract description 9
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims abstract description 5
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims abstract description 4
- 125000002091 cationic group Chemical group 0.000 claims abstract description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000003729 cation exchange resin Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 13
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- 230000002378 acidificating effect Effects 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
- 150000003863 ammonium salts Chemical class 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 239000003456 ion exchange resin Substances 0.000 claims description 4
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 125000004437 phosphorous atom Chemical group 0.000 claims description 4
- 229920001467 poly(styrenesulfonates) Polymers 0.000 claims description 4
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 4
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 4
- 239000011736 potassium bicarbonate Substances 0.000 claims description 4
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 4
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical group C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 2
- 125000004429 atom Chemical group 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 2
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 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 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 abstract description 2
- 229910019142 PO4 Inorganic materials 0.000 abstract 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 2
- 239000010452 phosphate Substances 0.000 abstract 2
- 239000000047 product Substances 0.000 description 34
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 17
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 17
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 17
- 239000001099 ammonium carbonate Substances 0.000 description 17
- 150000003833 nucleoside derivatives Chemical class 0.000 description 16
- 238000000926 separation method Methods 0.000 description 14
- 239000008367 deionised water Substances 0.000 description 11
- 229910021641 deionized water Inorganic materials 0.000 description 11
- 238000004679 31P NMR spectroscopy Methods 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- GUSFEBGYPWJUSS-UHFFFAOYSA-N pentaazanium;[oxido(phosphonatooxy)phosphoryl] phosphate Chemical compound [NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O GUSFEBGYPWJUSS-UHFFFAOYSA-N 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 8
- 238000001394 phosphorus-31 nuclear magnetic resonance spectrum Methods 0.000 description 8
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 229920005654 Sephadex Polymers 0.000 description 6
- 239000012507 Sephadex™ Substances 0.000 description 6
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 6
- 239000000843 powder Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- HWGNBUXHKFFFIH-UHFFFAOYSA-I pentasodium;[oxido(phosphonatooxy)phosphoryl] phosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O HWGNBUXHKFFFIH-UHFFFAOYSA-I 0.000 description 4
- 159000000000 sodium salts Chemical class 0.000 description 4
- 239000012043 crude product Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical class C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- ZRIUUUJAJJNDSS-UHFFFAOYSA-N ammonium phosphates Chemical compound [NH4+].[NH4+].[NH4+].[O-]P([O-])([O-])=O ZRIUUUJAJJNDSS-UHFFFAOYSA-N 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- KKTCWAXMXADOBB-UHFFFAOYSA-N azanium;hydrogen carbonate;hydrate Chemical compound [NH4+].O.OC([O-])=O KKTCWAXMXADOBB-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- AFQIYTIJXGTIEY-UHFFFAOYSA-N hydrogen carbonate;triethylazanium Chemical compound OC(O)=O.CCN(CC)CC AFQIYTIJXGTIEY-UHFFFAOYSA-N 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- MZQCFPABNHFYAT-UHFFFAOYSA-M sodium;propan-2-one;iodide Chemical compound [Na+].[I-].CC(C)=O MZQCFPABNHFYAT-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229940035637 spectrum-4 Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
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Abstract
Purification of nucleoside triphosphate derivatives is improved in yield rate. It is carried out by: reacting nuleoside phosphate with dichlorophosphate derivative to obtain a treated liquid containing the target product, purifying it with anionic exchange resin to obtain a mixed product containing phosphate and triphosphate, separating them from each other with cationic exchange resin, necessarily, using hydrogen carbonate to preparing triphosphate directly. The target product yields above 6 times.
Description
The technical field is as follows:
the invention relates to a purification method of nucleotide derivatives, in particular to a method for purifying and preparing nucleoside triphosphate derivatives.
Background art:
since nucleotides are dissociable compounds and the main part thereof is anions, they are usually separated and purified by anion exchange resins such as 717 and 201 or weakly basic anion exchange resins such as DEAE-Sephadex.
For synthetic nucleotide derivatives, separation and purification are usually also carried out using corresponding anion exchange resins.
The literature (J.Med.chem.1999, 42: 213) reports the following reactions of nucleoside monophosphate derivatives and dichlorophosphate derivatives:
the target product is a nucleoside triphosphate derivative. After the reaction is finished, DEAE-Sephadex anion exchange resin is adopted for reaction treatment liquid containing the target product nucleoside triphosphate derivative, triethylamine bicarbonate or ammonium bicarbonate water solution is used as eluent for separation and purification, the obtained nucleoside triphosphate ammonium salt is easy to absorb moisture, and sodium iodide acetone solution is used for converting the nucleoside triphosphate ammonium salt into sodium salt. As a result, the yield of the purified nucleoside triphosphate derivative of the object product is low, usually about 8%.
The main reason for this is that the unreacted nucleoside monophosphate derivative and the target nucleoside triphosphate derivative cannot be separated efficiently and completely by using the anion exchange resin alone, the cross-over fraction formed by the starting material and the product is large, and the fraction containing the pure product is small, so that the loss is large. Even if the cross-cut fraction is subjected to another anion exchange resin separation, the effect is very slight, and the raw materials and the products cannot be completely and effectively separated. In addition, in the process of converting the pure nucleoside triphosphate derivative ammonium salt which is separated from the anion exchange resin and is easy to absorb moisture into sodium salt by using an acetone solution of sodium iodide, the conversion rate is not high, and the crystallization is greatly reduced.
The invention content is as follows:
the technical problems to be solved by the invention are as follows: the purification yield of the synthetic nucleoside triphosphate derivative is improved; optimizing the purification method of the nucleoside triphosphate derivative.
The idea of the invention is as follows: the invention combines all mixed cross parts containing products obtained by separating with anion exchange resin, and then uses strong acid cation exchange resin to carry out ion exchange, so that unreacted raw material nucleoside monophosphate derivatives can be completely intercepted, target product nucleoside triphosphate derivatives are exchanged into original acid type on the resin and flow down without loss, and ammonium salts of the purified nucleoside triphosphate derivatives can be directly converted into corresponding adenosine triphosphate salts by using bicarbonate if necessary.
The technical scheme of the invention is as follows: a process for preparing nucleoside triphosphate derivative by purifying includes such steps as reaction between nucleoside monophosphate derivative and dichlorophosphate derivative to obtain the reaction liquid containing nucleoside triphosphate derivative, purifying by anionic exchange resin to obtain the mixed fraction containing nucleoside monophosphate and triphosphate, and purifying by cationic exchange resin to obtain purified nucleoside triphosphate derivative.
The strongly acidic cation exchange resin related to the invention is Dowex 50wx and corresponding type strongly acidic ion exchange resin, and for the separated and purified nucleoside triphosphate derivative with the molecular length of more than 20A, D001 with Dowex 50wx large pore diameter or corresponding type strongly acidic ion exchange resin is preferably used.
The bicarbonate of the invention is sodium bicarbonate or potassium bicarbonate.
The nucleoside triphosphate derivative has the general formula:
in the above formula:
b is a purine or pyrimidine base, and may be another nitrogen-containing heterocycle.
P is a group consisting of three phosphorus atoms, and two adjacent phosphorus atoms can be connected through other atoms besides the oxygen atom to form phosphoric anhydride.
Before the method is implemented, the reaction is well done, insoluble impurities in reaction liquid are removed by a filtering method after the reaction is finished, organic solvent is pumped out, dissolved by water, put on anion exchange resin, washed by water, then bicarbonate or ammonium bicarbonate solution of organic amine with gradientconcentration is used for washing away inorganic matters and organic matters, and fractions containing products are combined.
The invention is very simple to carry out by first converting the strongly acidic cation exchange resin to H+Form, the mixed cross-section containing product and starting material is combined and loaded such that the starting nucleoside monophosphate is trapped on the resin and the cation of the product nucleoside triphosphate is held by the H groups on the resin+After exchange, the solution flows into a container containing ammonium bicarbonate in the form of acid to reform ammonium salt, and a purification process is completed. Drying by a high vacuum freeze dryer, and completely removing the redundant ammonium bicarbonate to obtain the pure target product ammonium salt.
If the pure product of the obtained derivative ammonium salt is easy to absorb moisture, according to the invention, the pure product can be directly converted into corresponding sodium salt or potassium salt by using sodium bicarbonate or potassium bicarbonate with accurate equivalent:
(Note: ATP represents nucleoside triphosphate derivative)
The invention has the beneficial effects that: by adopting the method, the purification preparation yield of the target product nucleoside triphosphate derivative is improved by more than 6 times compared with the known literature method.
Description of the drawings:
FIG. 1 shows nucleoside ammonium triphosphates after anion exchange resin separation of example 11H-NMR spectrum
FIG. 2 shows nucleoside ammonium triphosphates afteranion exchange resin separation in example 131P-NMR spectrum
FIG. 3 shows the impurities separated by the anion exchange resin of example 11H-NMR spectrum
FIG. 4 shows the impurities separated by the anion exchange resin of example 131P-NMR spectrum
FIG. 5 shows nucleoside ammonium triphosphates after separation on cation exchange resin of example 11H-NMR spectrum
FIG. 6 shows nucleoside ammonium triphosphates after separation on cation exchange resin of example 131P-NMR spectrum
FIG. 7 nucleoside triphosphate sodium salt of example 11H-NMR spectrum
FIG. 8 is example 1 nucleoside triphosphate sodium salt31P-NMR spectrum
FIG. 9 shows crude anion exchange resin of example 2 after separation1H-NMR spectrum
FIG. 10 shows the crude product of example 2 after separation on anion exchange resin31P-NMR spectrum
FIG. 11 shows the recovery of nucleoside monophosphates after separation on the cation exchange resin of example 21H-NMR spectrum
FIG. 12 shows the recovery of nucleoside monophosphates after separation on the cation exchange resin of example 231P-NMR spectrum
FIG. 13 shows nucleoside ammonium triphosphates after separation on cation exchange resin of example 21H-NMR spectrum
FIG. 14 depicts nucleoside ammonium triphosphates after separation on cation exchange resin of example 231P-NMR spectrum
FIG. 15 is example 2 nucleoside triphosphate sodium salt1H-NMR spectrum
FIG. 16 is the nucleoside ammonium triphosphate salt after cation exchange resin separation of example 331P-NMR spectrum
The specific implementation mode is as follows:
example 1:
the reaction formula is as follows:
185 mg of tri-n-butylamine are dissolved in 15 ml of pyridine, and added583 mg of nucleoside monophosphate are dissolved by stirring and then dried under high vacuum at 40 ℃ and the vacuum is released under nitrogen. 12 ml of anhydrous DMF was added, and after dissolution, 810 mg of CDI was added and stirred overnight. Adding 246 mg of methanol, stirring for one hour, then dropwise adding a solution of 1392 mg of dichlorophosphoric acid and 1056 mg of tri-n-butylamine dissolved in 35 ml of DMF, stirring for reaction for 26 hours at room temperature, filtering, removing the DMF under high vacuum at the temperature of below 60 ℃, adding 20 ml of deionized water, and separating by an anion exchange resin DEAD-Sephadex A-100 column. Washing with 500 ml of deionized water, followed by washing with 250 ml of 0.1M ammonium bicarbonate and 250 ml of 0.2M ammonium bicarbonate to remove unreacted dichlorophosphoric acid, and finally eluting with 0.3M ammonium bicarbonate. During elution, the product was monitored by UV and once eluted, one portion per 25 ml. After freezing, respectively drying in vacuum freeze drier with H1NMR and P31NMR detection, the same fractions being pooled. 35 mg of pure product nucleoside triphosphate ammonium salt is obtained, the yield is 3.89 percent, and a spectrogram 1(1H-NMR) and spectrum 2(31P-NMR); 474 mg of impurity is obtained, see spectrogram 3(1H-NMR) and spectrum 4: (31P-NMR), from the phosphorus spectrum, approximately 14% (mol/mol) of unreacted nucleoside ammonium monophosphate was contained (single peak of. delta. 3.495).
Taking 20 g of pretreated macroporous strong-acid cation exchange resin D001 (H)+Type) was loaded onto the column and 474 mg of the white powder obtained above was dissolved in 50 ml of deionized water, the sample was loaded, the flow rate was controlled and the distillate was slowly run off, and when the distillate was acidic and had UV absorption, it indicated that the product nucleoside triphosphate had distilled off. The distillate containing the product flows directly intoA container containing 320 mg of ammonium bicarbonate. When the sample level dropped to the top of the resin, the washing with water continued until the distillate was neutral or free of UV, indicating that the product had distilled off. The nucleoside monophosphate remains in the resin, while the nucleoside triphosphate ammonium salt is identical to H+The cation exchange resin is ion exchanged and distilled out in the form of ortho, and the ortho reacts with ammonium bicarbonate to form ammonium salt. After the distillate is frozen, the distillate is placed in a vacuum freeze dryer for drying, and the excessive ammonium bicarbonate is sublimated to be full. 403 mg of pure nucleoside ammonium triphosphate are obtained, which are separated by anion exchange resin DEAD-Sephadex A-10035 mg of pure nucleoside ammonium triphosphate, total 438 mg, yield 49.29%, from spectrum 5 (R) (product X)1H-NMR) and spectrum 6(31P-NMR) that there was no trace of nucleoside monophosphate.
The obtained nucleoside triphosphate derivative ammonium salt is easy to absorb moisture, and can be converted into a sodium salt by using sodium bicarbonate with equivalent weight: 438 mg (0.497mmol) of the obtained pure nucleoside triphosphate ammonium salt was dissolved in a solutioncontaining 125 mg of sodium hydrogencarbonate (1.491mmol) and 10 ml of purified water, and after freezing, it was dried in a vacuum freeze-drying machine to obtain 445 mg of pure nucleoside triphosphate sodium salt as a white powder. See spectrum 7: (1H-NMR) and spectrum 8: (31P-NMR)。
Example 2:
the 2006 mg of thoroughly dried ammonium nucleoside monophosphate salt is dissolved in 10 ml of anhydrous pyridine, 665 mg of tri-n-butylamine are added, after dissolution, suction is carried out under reduced pressure, after which the oil pump is continued to completely remove the water, and the vacuum is released with nitrogen. 35 ml of absolute anhydrous DMF were added and 2718 mg of CDI were added after dissolution. After stirring for 14 hours, 936 mg of anhydrous methanol was added. After two hours, a solution of 4893 mg of dichlorophosphoric acid and 3694 mg of tri-n-butylamine in 120 ml of absolute anhydrous DMF was added dropwise and the reaction was stirred at room temperature for 24 hours. Filtering, pumping DMF, adding 100 ml deionized water, and separating with weak base anion exchange resin DEAD-Sephadex A-50 column. The solution was washed with 2000 ml of deionized water, then with 1000 ml of 0.1M ammonium bicarbonate and 1000 ml of 0.2M ammonium bicarbonate to wash out unreacted dichlorophosphoric acid, and finally eluted with 0.3M ammonium bicarbonate. During the elution process, the product is checked by UV, the fraction containing the product is frozen and then dried in a vacuum freeze dryer, and 2089 mg of crude product is obtained, and the product is shown in a graph 9 (A) (B)1H-NMR) and spectrum 10(31P-NMR) shows the presence of nucleoside monophosphate, containing about 18% (mol/mol) of nucleoside monophosphate31P-NMR)。
Dissolving the crude white powder in 200 ml of deionized water, and pre-treating into H+Type 100 g of macroporousstrongly acidic cation exchange resin D001 was packed in a column. The sample was loaded, the flow rate was controlled to slowly flow out, the product nucleoside triphosphate was monitored by UV for distillation, and the distillate was directly poured into a container containing 2000 mg of nucleoside triphosphateAnd (4) continuously washing with deionized water in the container of ammonium bicarbonate until no product is distilled. (after the product was distilled off, the nucleoside monophosphate remained in the resin, and was eluted with 2M aqueous ammonia, and the fraction containing the nucleoside monophosphate was frozen and then lyophilized under vacuum to recover 382 mg of the starting nucleoside monophosphate, as shown in FIG. 11:1H-NMR and Pattern 12:31P-NMR). After freezing the distillate containing the product nucleoside triphosphate, the product was dried in a vacuum freeze dryer to obtain 1622 mg of pure nucleoside triphosphate ammonium salt with a yield of 53.45%. See map 13(1H-NMR) and spectrum 14(31P-NMR). The pure nucleoside ammonium triphosphate salt is dissolved in 50 ml of pure water containing 463 mg of sodium bicarbonate, and the solution is frozen and then dried in a vacuum freeze dryer to obtain 1650 mg of white nucleoside sodium phosphate salt. See map 15(1H-NMR). Elemental analysis: c17H22Cl2F3N5Na3O12P3S2·4H2O, 912.94, C: calculated 22.33%, experimental 22.66%.
Example 3:
the reaction formula is as follows:
2.62 g of nucleoside monophosphate ammonium salt and 1.3 g of tri-n-butylamine were added to 20 ml of anhydrous pyridine, stirred to dissolve, then dried under high vacuum at 40 ℃and the vacuum was released under nitrogen. 70 ml of anhydrous DMF and 5.44 g of CDI were added. 1.88 g of anhydrous methanol was added with stirring overnight. A solution of 9.8 g of dichlorophosphoric acid and 7.4 g of tri-n-butylamine in 240 ml of anhydrous DMF was added dropwise with stirring, and the reaction was stirred at room temperature for one day. Filtering to remove white solid, pumping DMF by an oil pump, adding 200 ml of deionized water, stirring, pouring into a weak base anion exchange resin DEAD-Sephadex A-50 column, separating, washing with 4000 ml of deionized water, washing with 2000 ml of 0.1M ammonium bicarbonate and 2000 ml of 0.2M ammonium bicarbonate respectively, and washing with 0.3M ammonium bicarbonate. The elution process was monitored by UV, and the product containing fractions were frozen and dried in a vacuum freeze dryer to give 3250 mg of crude product. The crude white powder was dissolved in 300 ml of deionized water. Get anotherIs pretreated into H+Type 200 g of macroporous strongly acidic cation exchange resin D001 was packed in a column. And (3) controlling the outflow speed, monitoring whether the product nucleoside triphosphate is distilled off by using UV (ultraviolet), directly flowing the distillate into a container containing 4000 mg of ammonium bicarbonate, and continuously washing by using deionized water until no product is distilled off. After freezing the distillate containing the product nucleoside triphosphate, the product was dried in a vacuum freeze dryer to obtain 2520 mg of pure nucleoside triphosphate ammonium salt white powder with a yield of 58.79%. See map 16: (31P-NMR)。
Elemental analysis: c11H25Cl2N8O12P3·4H2O, 696.06, C: calculated value 18.95%, experimental value 19.02%;
h: calculated 4.77% and experimental 4.72%.
Claims (6)
1. A process for preparing nucleoside triphosphate derivative by purifying includes such steps as reaction between nucleoside monophosphate derivative and dichlorophosphate derivative to obtain the reaction liquid containing nucleoside triphosphate derivative, purifying by anionic exchange resin to obtain the mixed fraction containing nucleoside monophosphate and triphosphate, and purifying by cationic exchange resin to obtain purified nucleoside triphosphate derivative.
2. A process as claimed in claim 1, wherein the cation exchange resin involved is preferably Dowex 50wx and corresponding strongly acidic ion exchange resins.
3. The process as claimed in claim 1 or 2, wherein the isolated and purified nucleoside triphosphate derivatives having a molecular length of greater than 20A are preferably D001 with a Dowex 50wx large pore size or a corresponding type of strongly acidic ion exchange resin.
4. A process according to claim 1, characterised in that the bicarbonate is sodium bicarbonate or potassium bicarbonate.
5. The method as claimed in claim 1, wherein the purified ammonium salt of nucleoside triphosphate derivative is dissolved in water together with an equivalent amount of sodium bicarbonate or potassium bicarbonate, and the resulting solution is lyophilized under vacuum to give the corresponding sodium or potassium salt which is not deliquescent.
6. A method according to claim 1, wherein the nucleoside triphosphate derivative is:
in the above formula:
b is purine or pyrimidine base, and can also be other nitrogen heterocyclic ring;
p is a group consisting of three phosphorus atoms, and two adjacent phosphorus atoms can be connected through other atoms besides the oxygen atom to form phosphoric anhydride.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447927A (en) * | 2013-09-13 | 2015-03-25 | 天津市汉康医药生物技术有限公司 | Cangrelor monohydrate crystal and preparation method thereof |
| CN104447928A (en) * | 2013-09-13 | 2015-03-25 | 天津市汉康医药生物技术有限公司 | Cangrelor dihydrate |
| WO2019114674A1 (en) | 2017-12-12 | 2019-06-20 | 亚宝药业集团股份有限公司 | Method for prepare cangrelor tetrasodium salt |
| CN114736260A (en) * | 2022-03-29 | 2022-07-12 | 上海吉量医药工程有限公司 | Preparation method of nucleotide triphosphate |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5248998B2 (en) * | 1973-01-12 | 1977-12-14 | ||
| JPS518292A (en) * | 1974-07-13 | 1976-01-23 | Oriental Yeast Co Ltd | Adenoshin jifuosufueetono seiseiho |
| JPS5659795A (en) * | 1979-10-22 | 1981-05-23 | Kanegafuchi Chem Ind Co Ltd | Purification of nicotinic acid amide adenine dinucleotide |
| CN1147501C (en) * | 2000-12-15 | 2004-04-28 | 成都国嘉生物制药股份有限公司 | Process for preparing nucleotide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447927A (en) * | 2013-09-13 | 2015-03-25 | 天津市汉康医药生物技术有限公司 | Cangrelor monohydrate crystal and preparation method thereof |
| CN104447928A (en) * | 2013-09-13 | 2015-03-25 | 天津市汉康医药生物技术有限公司 | Cangrelor dihydrate |
| WO2019114674A1 (en) | 2017-12-12 | 2019-06-20 | 亚宝药业集团股份有限公司 | Method for prepare cangrelor tetrasodium salt |
| US11440934B2 (en) | 2017-12-12 | 2022-09-13 | Yabao Pharmaceutical Group Co., Ltd. | Method for preparing cangrelor tetrasodium salt |
| CN114736260A (en) * | 2022-03-29 | 2022-07-12 | 上海吉量医药工程有限公司 | Preparation method of nucleotide triphosphate |
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| CN100425619C (en) | 2008-10-15 |
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