WO2009129798A2 - Di- und triphosphat-prodrugs, insbesondere nukleosiddi- und -triphosphat-prodrugs - Google Patents
Di- und triphosphat-prodrugs, insbesondere nukleosiddi- und -triphosphat-prodrugs Download PDFInfo
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- WO2009129798A2 WO2009129798A2 PCT/DE2009/000550 DE2009000550W WO2009129798A2 WO 2009129798 A2 WO2009129798 A2 WO 2009129798A2 DE 2009000550 W DE2009000550 W DE 2009000550W WO 2009129798 A2 WO2009129798 A2 WO 2009129798A2
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- nucleoside
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- 0 *C(Oc1ccc(COP(OCc(cc2)ccc2O)(OP(*)(O)=O)=O)cc1)=O Chemical compound *C(Oc1ccc(COP(OCc(cc2)ccc2O)(OP(*)(O)=O)=O)cc1)=O 0.000 description 10
- OJPNKYLDSDFUPG-UHFFFAOYSA-N C=C(C=C1)C=CC1=O Chemical compound C=C(C=C1)C=CC1=O OJPNKYLDSDFUPG-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/242—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyaryl compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
Definitions
- Di- and triphosphate prodrugs in particular nucleoside di- and triphosphate prodrugs
- the invention relates to compounds which can be used as prodrugs, medicaments and pharmaceutical compositions obtainable therefrom, as well as pharmaceutical dosage forms and a process for the preparation of the compounds according to the invention.
- nucleoside analogues that can be incorporated into the DNA.
- AIDS immunodeficiency Syndrome
- HI virus which belongs to the group of retroviruses.
- forms of therapy are known which suppress the proliferation of the virus.
- not a single drug is known that could completely eliminate the virus from the body.
- the medication leads in some patients to unpleasant side effects [D. Richman, M.A. Fischl, M.H. Grieco, M.S. Gott Kunststoff, P.A. Volberding, O.L. Laskin, J.M. Leedom, J. Groopman, D.Mildvan, M.S. Hirsch, G.G. Jackson, DT. Durack, D. Phil, S.
- Natural nucleosides as well as antivirally active nucleoside analogs must first be converted into their triphosphates by certain, more or less specific enzymes when they are incorporated into the DNA strand for chain extension.
- the structure of the NTP from the corresponding nucleoside analogue is carried out by kinases via three individual steps (nucleoside-> nucleoside monophosphate (NMP) ->NDP-> NTP), each of which inhibited or not expire because of the structural variation of the nucleoside analog compared to the parent nucleoside can.
- NMP nucleoside monophosphate
- NTP nucleoside triphosphate
- the antivirally active nucleoside analogs act as chain terminators, ie there is no further elongation in the 3 'direction [J. Baizarini, P. Herdewijn, E. De Clercq; Differential Patterns of intracellular Metabolism of 2 ', 3'-didehydro-2', 3'-dideoxythymidine and 3, 3 '-dideoxythymidine' -azido-2 ', two potent anti-human immunodeficiency virus Compounds; J. Biol. Chem. 1989, 264, 6127-6133].
- BBB blood-brain barrier
- prodrug systems One way to increase the lipophilicity of known drugs is the use of prodrug systems.
- Prodrugs or "prodrugs” are prodrugs which release the actual active ingredient later with elimination of masking groups.
- Prerequisites for such a prodrug, in this case a pronucleotide are increased lipophilicity in order to be able to penetrate the cell membranes and the blood-brain barrier, sufficient stability in the extracellular medium and the release of non-toxic masks.
- NMP prodrugs A disadvantage of the use of NMP prodrugs is further that the required further phosphorylation to the di- and triphosphates in the cell can be inhibited or completely prevented.
- DP diphosphate
- DP diphosphate
- DP diphosphate
- MP monophosphate
- NDP and NTP In contrast to NMP prodrugs, there is currently no functioning system for masking NDP and NTP.
- the application of the concepts developed for NMP prodrugs is described as not possible in the prior art [s. Chu and Tan 1999 (Advanced Drug Delivery Reviews)].
- the problem is that in the case of NDP and NTP, in contrast to NMP, no simple phosphate group, but rather a high-energy phosphoric anhydride bond in the form of the pyrophosphate unit has to be reversibly masked without the anhydride bond (s) breaking.
- anhydride bond s
- NDP and NTP prodrugs essentially from their NMP relatives.
- hydrolysis reactions can take place on the phosphorus atom. Since masking of a pyrophosphate bridge always results in one more charge than for a monophosphate, it is not expected that enzymatic cleavage of NDP or NTP prodrugs could be efficient. Rather, considering the prior art [C. Meier, U. Muus, J. Renze, L. Naesens, E. De Clercq, J.
- the object of the present invention is to provide improved di- and / or triphosphate prodrugs, in particular nucleoside or nucleoside analog prodrugs, which do not have the disadvantages known from the prior art.
- the invention relates in a first aspect to a compound of general formula (I)
- R 1 , R 4 , R 7 and R 8 independently of one another are H, OCR or HN-C-R, R is alkyl, aryl or O-acyl,
- R 2 , R 3 , R 5 and R 6 are independently H, alkyl, aryl or O-acyl, R 9 is a nucleoside, nucleoside analog or alcohol residue, and Kat + is a cation, provided that R 1 , R 4 , R 7 and R 8 are not all H.
- nucleoside is understood as meaning organic molecules which consist of a sugar residue and an organic base, in particular a nitrogen-containing heterocyclic base (nucleobase).
- the sugar residue is usually a pentose, e.g. Deoxyribose or ribose.
- the nucleobases are often purines (R) and pyrimidines (Y). Examples of purines are guanine (G) and adenine (A), examples of pyrimidines are cytosine (C), thymine (T) and uracil (U).
- Phosphorylated nucleosides for example nucleoside monophosphates (NMP), nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP), are also referred to as nucleotides.
- NMP nucleoside monophosphates
- NDP nucleoside diphosphates
- NTP nucleoside triphosphates
- the phosphate, diphosphate (pyrophosphate) or triphosphate group is usually linked to the 5'-C atom of the sugar component of the nucleoside.
- a nucleoside analog is here understood to mean a nucleoside which naturally does not occur in the human body but resembles a naturally occurring nucleoside in the human body in such a way that it is processed by the cell in accordance with the natural nucleoside, for example phosphorylated and incorporated into a DNA strand
- alkyl includes saturated aliphatic groups including straight-chain alkyl groups (eg, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl), branched chain alkyl groups (eg, isopropyl, tert-butyl, isobutyl), cycloalkyl (eg, alicyclic ) Groups (eg cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl-substituted cycloal
- Alkyl further includes alkyl groups having oxygen, nitrogen, sulfur or phosphorus atoms which replace one or more carbon atoms of the hydrocarbon backbone.
- alkyl also includes both unsubstituted alkyls and substituted alkyls, the latter referring to alkyl groups having substituents replacing a hydrogen atom on one or more carbon atoms of the hydrocarbon backbone.
- substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano , Amino (including alkylamino, dialkylamino,
- Arylamino, diarylamino and alkylarylamino may be acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl , Alkylaryl or an aromatic or heteroaromatic radical.
- Cycloalkyls may be further substituted, eg with the substituents given above.
- An "alkylaryl” or “aralkyl” radical is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
- “Alkyl” also includes the side chains of natural and unnatural amino acids.
- aryl is meant groups having aromaticity, including 5- and 6-membered single-ring aromatic groups which may include from zero to four heteroatoms, as well as multi-cyclic systems having at least one aromatic ring.
- aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
- aryl includes multicyclic aryl groups, eg tricyclic, bicyclic, eg naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine or indolizine ,
- aryl are also meant aryl groups which have heteroatoms in the ring structure (“heteroaryls").
- the aromatic ring may be substituted at one or more ring positions.
- Aryl groups may also contain alicyclic or heterocyclic rings that are not aromatic are fused or bridged to form a multicyclic system (eg tetralin, methylenedioxyphenyl).
- alcohol radical is understood here to mean any organic carbon compound in which a hydrogen atom on the carbon skeleton is replaced by a hydroxyl group.
- alcohols include sugars such as glucose, fructose, mannose etc, but also other compounds such as e.g. Geraniol.
- the compounds of the invention can be used in an advantageous manner as prodrugs (prodrugs). They allow for the first time the introduction of nucleosides and nucleoside analogs and other organic compounds having a hydroxyl group, e.g. of sugars or alcohols, at the level of di- and triphosphates in the cell. This makes it possible for the first time, for example, to introduce the nucleoside triphosphates used by the cell directly into the cell so that any further intracellular phosphorylation steps that are otherwise required are no longer needed. As a result, not only can the actual use of the introduced nucleosides by the cell be made better possible, but also side effects, which are caused, for example, by monophosphates, can now be avoided.
- the compounds according to the invention can find advantageous use as medicaments both as antiviral and as antitumor agents. They are particularly suitable as medicines for
- Non-nucleosidic alcohols can also be transported into the cell in the form of their di- or triphosphates and released there.
- the lipophilicity of the compounds according to the invention can be varied and adjusted. In this way it is possible to tailor the connections for different applications.
- the skilled worker knows of methods by which he can introduce the corresponding residues and determine the lipophilicity.
- R 1 , R 4 , R 7 and R 8 are simultaneously hydrogen atoms. However, this does not exclude that, for example, R 1 , R 4 and R 7 or R 1 , R 7 and R 8 or R 4 , R 7 and R 8 are all H. In a preferred embodiment, in the compound of the invention R 1 and
- R 4 both OCR or HN-C-R and R 7 and R 8 are both H.
- R 9 in the compound of the invention is a nucleoside or nucleoside analog.
- R 9 can also be any alcohol radical, the alcohol particularly preferably being an alcohol which is used in the cell in the form of a diphosphate or triphosphate.
- An example of such an alcohol is geraniol, which occurs in the biosynthesis of pheromones in the form of its diphosphate.
- Exemplary compounds according to the invention are bis (4-acyloxybenzyl) -nucleoside diphosphate (BAB-NDP) and bis (4-acyloxybenzyl) -nucleoside triphosphate (BAB-NTP) and the corresponding OAf ⁇ o isomers bis- (2-acyloxybenzyl) -nucleoside diphosphate (BAB-NDP) and bis (2-acyloxybenzyl) -nucleoside triphosphate:
- the compounds according to the invention are converted into bis (4-acylaminobenzyl) -nucleoside diphosphate, bis- ( ⁇ / -acylaminobenzyl) -nucleoside diphosphate, bis - ( ⁇ / -acylaminobenzyl) -nucleoside triphosphate and bis- (N-acylaminobenzyl) -nucleoside triphosphate:
- acyl radical R can be used to control the stability in various media and at the same time adjust the polarity of the compound so that effective passive transport across the cell membrane can be achieved. Masking of the non-terminal phosphates is omitted.
- the prodrugs according to the invention show a high stability to chemical hydrolysis in aqueous buffer systems and a clear tendency to enzymatic hydrolysis and thus release of the active ingredient in human cell extracts. They fulfill important requirements for use as pharmaceuticals.
- the hydrolysis mechanism of the BAB-N DP prodrug according to the invention is shown below:
- the invention also relates in a further aspect to a pharmaceutical
- a composition comprising a compound of the invention and a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers are known to those skilled in the art and include one or more liquid, semi-solid or solid fillers, diluents or other substances suitable for administration to mammals, including humans.
- carrier in the context of the present invention refers to any organic or inorganic, natural or synthetic substance which can be combined with the active ingredient to simplify the administration. Examples of such carriers include, but are not limited to, organic or inorganic solvents, starch, lactose, mannitol, methylcellulose, talc, gelatin, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats, higher molecular weight fatty acids, or higher molecular weight polymers.
- “Pharmaceutically acceptable” means any non-toxic material for mammals, particularly humans, that does not substantially affect the effectiveness of the biological activity of the active ingredient. Such materials may include pharmaceutically acceptable levels of salts, buffers, preservatives, or the like.
- Non-limiting examples of pharmaceutically acceptable carriers include magnesium carbonate, magnesium stearate, talc, sugars, lactose, ethanol, glycerine, water, etc.
- the pharmaceutical composition may also comprise adjuvants and / or diluents.
- the invention also relates to a pharmaceutical
- Dosage form comprising a compound of the invention and a pharmaceutically acceptable carrier.
- a dosage form for oral administration for example a tablet or capsule.
- the present invention relates to a process for the preparation of a compound of the general formula (I)
- R 1 , R 4 , R 7 and R 8 independently of one another are H, OCR or HN-C-R, R is alkyl, aryl or O-alkyl,
- R 2 , R 3 , R 5 and R 6 are independently H, alkyl, aryl or Oalkyl, R 9 is nucleoside, nucleoside analog or alcohol residue, and Kat + is a cation, provided that R 1 , R 4 , R 7 and R 8 are not all H, comprising the steps of a) reacting a phenyl ester of the general formula (III)
- R 4 is chosen so that both are O-CR or HN-C-R and R and R 8 are chosen so that both are H.
- R 7 and R 8 are chosen such that both are O-CR or HN-C-R and R 1 and R 4 are chosen so that both are H.
- R 9 is a nucleoside or nucleoside analog.
- Fig. 4 Stability of the prodrugs of the invention in human blood plasma.
- Phenylester IM are reacted with Dichlorophosphoramidit 194, preferably under inert gas (eg nitrogen), with cooling (eg -78 0 C) and in abs. Triethylamine (TEA) dissolved in abs. Tetrahydrofuran (THF).
- THF Tetrahydrofuran
- the resulting phosphoramidite IV is then reacted with a corresponding nucleoside monophosphate (NucIMP) or nucleoside diphosphate (NucIDP) to form the corresponding nucleoside di (Ia) and triphosphates (IIa), respectively.
- the corresponding NTP prodrugs can be prepared by using the corresponding nucleoside diphosphates instead of the nucleoside monophosphates (NucIMP).
- the synthesis of all compounds I and II according to the invention can be carried out according to the method described on the example of the BAB-NDP prodrugs.
- 4-hydroxybenzyl alcohol 21 is selectively acylated with the corresponding acid chlorides.
- the reactions are carried out under nitrogen as an inert gas. It will be 1.0 eq. p-hydroxybenzyl alcohol and 1.0 eq. Section. Triethylamine (TEA) in abs. Dissolved tetrahydrofuran (THF) and cooled to 0 0 C. For 20 minutes, the respective acid chlorides (1.1 eq.), Dissolved in abs. THF, dripped. After 1-2 h reaction time at 0 0 C triethylammonium chloride is removed by filtration and condensed off the solvent in vacuo.
- TFA Triethylamine
- Phosphorus trichloride is reacted with diisopropylamine DIPA in diethyl ether. This is followed by Schlenk filtration. The result is dichlorophosphoramidite (194).
- Variant B The reactions are carried out under nitrogen as an inert gas. It will be 4.4 eq. POCI 3 in abs. Dissolved acetonitrile and cooled to 0 ° C. This solution will be 4.4 eq. Section. Pyridine and 2.2 eq. carefully added distilled water. After 5-10 min, 1.0 eq. the relevant nucleoside added as a solid. After 4 h at RT (room temperature) temperature), the reaction is terminated by adding ice water and stirred for a further hour in the refrigerator. By careful addition of solid ammonium hydrogen carbonate, a pH of 8 is set. The solvents are removed by freeze-drying. The residue is dissolved in water and purified on an RP-18 phase with water and an acetonitrile gradient.
- Variant A The NMPs are eluted through a protonated ion exchange column Dowex 50WX8.
- the protonated form of the NMP is titrated with the respective cationic hydroxide solution to the neutral point or by direct addition of 2.0 eq. neutralized the respective solutions and the solvent removed by lyophilization.
- Variant B The diammonium salts of NMPs are dissolved in water. It will be 2.0 eq. Tetra-n-butylammonium hydroxide solution (40% in H 2 O 1 m / m) was added and the solvent removed by freeze-drying.
- the Solution is cooled to -25 0 C and oxidized with 51 ul f-BuOOH (5.5 M in n-decane, 0:28 mmol, 1.4 eq.). After 15 min at -25 0 C is warmed to RT and the solvent removed in vacuo.
- the crude product is purified on a RP-18 column, first with water / methanol 2: 1, then 1: 1.
- the product is lyophilized.
- the counterions are exchanged (Dowex 50WX8, NH 4 + ).
- the eluate is lyophilized.
- the product is again chromatographed RP-18, first with water / methanol 2: 1, then 1: 1.
- the product is again freeze-dried. Yield: 14 mg (20 ⁇ mol, 10%) of a colorless, hygroscopic solid.
- the mixture is then cooled to -25 0 C and quenched by addition of 46 ⁇ l_ f-BuOOH (5.5 M in ⁇ -decane, 0:25 mmol, 1.0 eq.) Was oxidized. After 15 min at -25 0 C is warmed to RT and the solvent removed in vacuo.
- the crude product was purified via an RP-18 reversed phase (water / methanol, initially 2: 1, then 1: 1, then pure methanol). The product fractions are lyophilized.
- the crude product undergoes ion exchange (Dowex 50WX8, NH 4 + ).
- the eluate is lyophilized.
- a final purification is performed on an RP-18 reverse phase (water / methanol 1: 2). The product is freeze-dried.
- the reaction is carried out under nitrogen as an inert gas.
- 115 mg of bis (tetra-n-butylammonium) AZTMP (139 ⁇ mol, 1.0 eq.) are initially suspended for 2 h over molecular sieve 0.3 nm in 3 mL abs.
- Dried acetonitrile The dried solution is transferred to a reaction flask and the solvent removed in vacuo.
- the molecular sieve is washed twice with abs. Washed acetonitrile and the wash solution also transferred to the reaction flask.
- the educt is still three times with a few mL of abs.
- Acetonitrile coevaporated to give a colorless foam. This is in 1.5 mL abs.
- Composition NBu 4 ZNH 4 1: 3 is obtained.
- the NMR data are given. Renewed ion exchange is dispensed with since about 7% decomposition product occurred in the previously pure product with a single ion exchange.
- Variant A The reaction is carried out under nitrogen as an inert gas. 146 mg of bis (tetra-n-butylammonium) AZTMP (176 ⁇ mol, 1.0 eq.) Are initially suspended for 2 h over molecular sieve 0.3 nm in 3 mL abs. Dried acetonitrile. The dried solution is transferred to a reaction flask and the solvent removed in vacuo. The molecular sieve is washed twice with abs. Washed acetonitrile and the wash solution also transferred to the reaction flask. The educt is still three times with a few mL of abs. Acetonitrile coevaporated to give a colorless foam. This is in 2 mL abs.
- the resulting fractions are always lyophilized on the lyophil.
- the pure product obtained is re-salted on an ion exchanger (Dowex 50WX8, NH 4 + ) and lyophilized. A proportionate decomposition (about 7%) of the product occurred.
- Variant B The reaction is carried out under nitrogen as an inert gas. There are 26 mg of di-ammonium AZTMP (68 .mu.mol, 1.0 eq.) Twice with abs. Acetonitrile coevaporated, the residue in 3 ml abs. DMF was added and a few beads of molecular sieve 0.4 nm added.
- the oxidation was performed by adding 22 .mu.l NBuOOH (5.5 M in n-decane, 0:12 mmol, 1.8 eq.) At -30 0 C.
- the solvents are removed in vacuo, the residue taken up in water / acetonitrile and lyophilized. From the crude product, a 31 P-NMR spectrum is recorded. Although the product was formed, it was found that variant A gave significantly better yields with fewer by-products. On a separation of the crude mixture is therefore omitted.
- reaction is carried out under nitrogen as an inert gas.
- 160 mg of bis (tetra-n-butylammonium) d4TMP (203 ⁇ mol, 1.0 eq.) are initially suspended for 2 h over molecular sieve 0.3 nm in 3 mL abs.
- Dried acetonitrile The dried solution is transferred to a reaction flask and the solvent removed in vacuo.
- the molecular sieve is washed twice with abs. Washed acetonitrile and the wash solution also transferred to the reaction flask.
- the educt is still three times with a few mL of abs. Acetonitrile coevaporated to give a colorless foam. This is in 2 mL abs.
- the crude product is dissolved in a little methanol and separated on a RP-18 phase, first isocratically with water / methanol 1: 1 to remove unreacted starting material and DCI, then isocratically with water / methanol 5: 1.
- the product fractions are lyophilized and the crude product is added via an ion exchanger (Dowex 50WX8, NH 4 + , 30% acetonitrile in water).
- the eluate is lyophilized again and the residue is re-separated with water / methanol 1: 4 on an RP-18 phase.
- the product is finally lyophilized.
- the work-up is carried out as follows: After completion of the reaction, the solvent is removed in vacuo and the residue is dissolved in a little acetonitrile. This solution is chromatographed isocratically on a RP-18 phase with water / methanol 1: 1 until educt and DCI have been removed. Now is eluted isocratically with water / methanol 3: 1 and then 4: 1. For both mixtures, the product could be detected.
- the clean fractions (F1) are combined and freed from methanol on a rotary evaporator at low temperature, made up with water and freeze-dried. There are obtained 58 mg of the clean fractions (F1) as tetra-n-butylammonium salt.
- the contaminated fractions (F2) are added twice via an ion exchanger (Dowex 50WX8, NH 4 + , 30% acetonitrile in water), lyophilized and rechromatographed RP-18 (acetonitrile / water 2: 1).
- the resulting crude product (F2) is chromatographed on preparative HPLC (water / acetonitrile 1: 1, 10 mL / min, RP-18). With an applied amount of 5 mg, the product could be kept pure. With a larger amount, however, the breakup failed.
- the reaction is carried out under nitrogen as an inert gas.
- the drying of the nucleotide and the reaction procedure were carried out as described in the preceding experiments.
- the reaction is carried out under nitrogen as an inert gas.
- the drying of the nucleotide and the reaction procedure were carried out as described in the preceding experiments.
- the oxidation was performed by adding 42.0 .mu.l f-BuOOH (5.5 M in n-decane, 232 .mu.mol, 2.0 eq.) After 15 min at -25 0 C. the solvent was removed in vacuo. The residue is dissolved in a little acetonitrile and chromatographed on an RP-18 phase, first with methanol / water 1: 1, then 4: 1. Methanol is removed on a rotary evaporator, made up with water and lyophilized. The crude product is again separated via a RP-18 phase with methanol / water 2.5: 1.0. The product is lyophilized and chromatographed on preparative HPLC (40 mM ammonium formate in H 2 O / acetonitrile 1: 1, flow rate 10 ml / min, RP-18).
- the reaction is carried out under nitrogen as an inert gas.
- the drying of the nucleoside and the reaction were carried out as described in the preceding experiments.
- 50 mM stock solutions of the target compounds in DMSO are prepared. 11 ⁇ l of these stock solutions are diluted in each case with 189 ⁇ l of deionized water and a further 100 ⁇ l of DMSO, so that 1.9 mM hydrolysis stock solutions are obtained.
- the samples are incubated in a thermomixer at 37 ° C. After a period of time, aliquots of the kinetics solution are taken (Qe 60 ⁇ L) and immediately frozen in liquid nitrogen. The samples are thawed individually and analyzed immediately.
- 547 mg disodium hydrogen phosphate and 155 mg potassium dihydrogen phosphate are made up to 100 ml total volume with deionized water.
- the pH is controlled and optionally adjusted with phosphoric acid or NaOH.
- 30 ⁇ l of the 50 mM stock DMSO solution of the compounds are diluted to a concentration of 6.0 mM with 220 ⁇ l of DMSO.
- 100 ⁇ l of cell extract CEM / 0 for d4T, P3HR1 for BVdU and ACV, mouse liver extract for various compounds
- 20 ⁇ l of 70 mM magnesium chloride solution For each measurement, such a solution is used and incubated at 37 0 C in the thermomixer.
- the hydrolyses are quenched by the addition of 300 ⁇ L of methanol and stored at 0 ° C. for 5 min.
- the hydrolysis kinetics in culture medium were analogous to the kinetics in cell extracts. In contrast, however, no MgCl 2 solution is added. Instead of the cell extract, culture medium (RPMI) with 10% heat-inactivated fetal calf serum (FCS) is added.
- RPMI culture medium
- FCS heat-inactivated fetal calf serum
- AZT stands for the nucleoside analogue 3'-azido-2 ', 3'-dideoxythymidine, also known as retrovir.
- the NMR spectroscopic monitoring of the hydrolysis clearly shows the preferential formation of 186 from 185a. If any cleavage of the anhydride bond to AZTMP occurs, then in a minor degree.
- the assignment of the individual signals can be achieved for 185a by 31 P- 1 H correlation spectroscopy.
Abstract
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PCT/DE2009/000550 WO2009129798A2 (de) | 2008-04-24 | 2009-04-24 | Di- und triphosphat-prodrugs, insbesondere nukleosiddi- und -triphosphat-prodrugs |
Country Status (2)
Country | Link |
---|---|
DE (2) | DE102008020633A1 (de) |
WO (1) | WO2009129798A2 (de) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102014112055A1 (de) | 2014-08-22 | 2016-02-25 | Universität Hamburg | Di- und Triphosphat-Propharmaka |
WO2018100137A1 (en) | 2016-12-02 | 2018-06-07 | Universität Hamburg | Nucleoside triphosphate and nucleoside triphosphate analogue prodrugs |
EP4151646A1 (de) | 2021-09-20 | 2023-03-22 | Universität Hamburg | 5-fluoruracilderivate als prodrugs zur krebsbehandlung |
EP4356928A1 (de) | 2022-10-20 | 2024-04-24 | Universität Hamburg | Nukleosid-diphosphat- oder diphosphonat-prodrugs oder nukleosid-analog-diphosphat- oder diphosphonat-prodrugs |
-
2008
- 2008-04-24 DE DE200810020633 patent/DE102008020633A1/de not_active Withdrawn
-
2009
- 2009-04-24 DE DE112009001591T patent/DE112009001591A5/de not_active Withdrawn
- 2009-04-24 WO PCT/DE2009/000550 patent/WO2009129798A2/de active Application Filing
Non-Patent Citations (5)
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102014112055A1 (de) | 2014-08-22 | 2016-02-25 | Universität Hamburg | Di- und Triphosphat-Propharmaka |
WO2016026493A1 (de) | 2014-08-22 | 2016-02-25 | Universität Hamburg | Di- und triphosphat-propharmaka |
CN106661075A (zh) * | 2014-08-22 | 2017-05-10 | 汉堡大学 | 二磷酸和三磷酸前体药物 |
US10131685B2 (en) | 2014-08-22 | 2018-11-20 | Universitaet Hamburg | Di- and triphosphate prodrugs |
WO2018100137A1 (en) | 2016-12-02 | 2018-06-07 | Universität Hamburg | Nucleoside triphosphate and nucleoside triphosphate analogue prodrugs |
EP4151646A1 (de) | 2021-09-20 | 2023-03-22 | Universität Hamburg | 5-fluoruracilderivate als prodrugs zur krebsbehandlung |
WO2023041786A1 (en) | 2021-09-20 | 2023-03-23 | Universität Hamburg | 5-fluorouracil derivatives as prodrugs for cancer treatment |
EP4356928A1 (de) | 2022-10-20 | 2024-04-24 | Universität Hamburg | Nukleosid-diphosphat- oder diphosphonat-prodrugs oder nukleosid-analog-diphosphat- oder diphosphonat-prodrugs |
Also Published As
Publication number | Publication date |
---|---|
DE102008020633A1 (de) | 2009-10-29 |
WO2009129798A3 (de) | 2009-12-17 |
DE112009001591A5 (de) | 2011-04-07 |
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