DE102008020633A1 - Nucleoside di- and nucleoside triphosphate prodrugs - Google Patents

Abstract

The invention relates to a compound which can be used for 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. The invention provides bioreversible masked nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) as well as corresponding nucleoside analogs which are particularly useful in the treatment of viral diseases such as AIDS.

Description

The The invention relates to compounds which can be used as prodrugs, from them Preparable drugs and pharmaceutical compositions as well as pharmaceutical dosage forms and a process for the preparation the compounds of the invention.

In the treatment of viral infectious diseases and cancer it is promising to use nucleoside analogues which are in the DNA can be incorporated.

Among the most threatening infectious diseases is the immunodeficiency syndrome AIDS (Acquired Immunodeficiency Syndrome). Responsible for AIDS is the HI virus, which belongs to the group of retroviruses. Meanwhile, forms of therapy are known which suppress the proliferation of the virus. In fact, not a single drug is known that could completely eliminate the virus from the body. The medication also causes unpleasant side effects in some patients [ DD Richman, MA Fischl, MH Grieco, MS Gottlieb, PA Volberding, OL Laskin, JM Leedom, J. Groopman, D. Mildvan, MS Hirsch, GG Jackson, DT Durack, D. Phil, S. Nusinoff-Lehrman; The toxicity of AZT in the treatment of patients with AIDS and AIDS-related complex; New Engl. J. Med. 1987, 317, 192-197. ]. There is therefore an interest in finding new active ingredients and improving known ones in terms of their effectiveness and tolerability. In particular, the optimization of existing active ingredients brings some advantages, since these are already being studied in detail. If it is possible to dose a drug lower by optimizing the properties, the side effects should only occur in a weakened form.

natural Nucleosides as well as antivirally active Nukleosidanaloga must first by certain, more or less specific Enzymes are converted into their triphosphates, when incorporated into the DNA strand for chain extension become. The construction 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 because of structural variation of the nucleoside analogue compared to Stem nucleoside inhibited or can not occur. The nucleoside triphosphate (NTP) thus represents the active compound.

After incorporation, the antivirally active nucleoside analogs act as chain terminators, ie there is no further elongation in the 3 'direction [ Balzarini, P. Herdewijn, E. De Clercq; Differential Patterns of Intracellular Metabolism of 2 ', 3'-Didehydro-2', 3'-Dideoxythymidine and 3'-Azido-2 ', 3'-Dideoxythymidine, Two Potent Anti-human Immunodeficiency Virus Compounds; J. Biol. Chem. 1989, 264, 6127-6133 ]. The following are some examples of anti-HIV active nucleoside analogues. They all have in common the absence of the 3'-OH group.

Passing the blood-brain barrier (BBB) is a particular challenge in diseases that also affect the brain. It is therefore important to design drugs or modify them to be able to do so to pass the BBB. Some studies have shown that diffusion across the BBB increases with increasing lipophilicity of the drugs [ RJ Sawchuk, Z. Yang; Investigation of distribution, transport and uptake of anti-HIV drugs to the central nervous system; Advanced Drug Delivery Reviews 1999, 39, 5-31 ].

A Ability to increase the lipophilicity of known drugs, lies in the use of prodrug systems. Prodrugs or "prodrugs" are Active substance precursors, which the actual active substance only release later with elimination of masking groups. Requirements for such a prodrug, in this case a pronucleotide, are an increased lipophilicity to the Penetrating cell membranes and the blood-brain barrier, sufficient stability in the extracellular Medium and the release of non-toxic masks.

For NMP there are already some approaches for enzymatically activatable prodrug systems [ A. Pompon, I. Lefebvre, J.-L. Imbach, S. Khan, D. Farquhar; Decomposition Pathways of the Mono- (Pivaloyloxymethyl) and Bis- (Pivaloyloxymethyl) Esters of Azidothymidine 5'-Monophosphates in Cell Extract and in Tissue Culture Medium - An Application of the Online Isrp-Cleaning HPLC Technique; Antiviral Chem. Chemother. 1994, 5, 91-98 ; I. Lefebvre, C. Perigaud, A. Pompon, A.-M. Aubertin, J.-L. Girardet, A. Kim, G. Gosselin, J.-L. Imbach; Mono Nucleosides Phosphotriester Derivatives with S-Acyl-2-thioethyl Bioreversible Phosphate Protecting Groups: Intracellular Delivery of 3'-azido-2 ', 3'-dideoxythymidine-5'-monophosphates; J. Med. Chem. 1995, 38, 3941-3950 ; W. Thomson, D. Nicholls, WJ Irwin, JS Al-Mushadani, S. Freeman, A. Karpas, J. Petrik, N. Mahmood, AJ Hay, Synthesis, Bioactivation and Anti-HIV Activity of the Bis (4-acyloxybenzyl ) and mono (4-acyloxybenzyl) esters of the 5'-monophosphate of AZT, J. Chem. Soc., Perkin Trans., 1993, 1, 1239-1245 ]

Bis- (4-acyloxybenzyl) (AB) nucleotides were obtained from S. Freeman et al. described [ W. Thomson, D. Nicholls, WJ Irwin, JS Al-Mushadani, S. Freeman, A. Karpas, J. Petrik, N. Mahmood, AJ Hay, Synthesis, Bioactivation and Anti-HIV Activity of the Bis (4-acyloxybenzyl ) and mono (4-acyloxybenzyl) esters of the 5'-monophosphate of AZT, J. Chem. Soc., Perkin Trans., 1993, 1, 1239-1245 ; A. Routledge, I. Walker, S. Freeman, A. Hay, N. Mahmood, Synthesis, Bioactivation and Anti-HIV Activity of 4-acyloxybenzyl Bis (nucleoside-5-yl) Phosphates; Nucl. Nucl., 1995, 14 (7), 1545-1558 ]. In order to obtain a sufficient substrate affinity of carboxyesterases over the intermediately occurring phosphate diester, an aromatic system is introduced as a spacer. The hydrolysis cascade is initiated by cleavage of the acyl group of 17. This results in a reversal of the benzyl group para-permanent rest of the electron acceptor to an electric nendonator instead. This reversal labile stabilizes the benzyl ester linkage and results in spontaneous decay of intermediate 18 into the AB diester 19 and a mesomerically stabilized 4-quinone methide 20. Repeating the cascade releases the NMP. The cleavage mechanism of bis (AB) nucleotides is shown below (Nucl = nucleoside):

in the Case of NMP must have two negatively charged oxygen atoms be masked lipophilic. It turns out that the cleavage of the acyl radicals by esterases for the cleavage of the second mask clearly is slowed down. This will be due to the resulting negative charge Cleavage of the first mask returned.

adversely the use of NMP prodrugs continues to be that required further phosphorylation to the di- and triphosphates in the cell can be inhibited or completely prevented. So is for example, in the case of AZT, a known anti-HIV drug, the phosphorylation to AZTDP (DP = diphosphate) inhibited. About that In addition, numerous side effects become the corresponding monophosphate Attributed to AZTMP (MP = monophosphate).

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. When unmasking the pyrophosphate moiety, there must be no reaction at the phosphorus atom, as this may lead to breakage of the pyrophosphate bridge. This distinguishes NDP and NTP prodrugs essentially from their NMP relatives. Here also 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 state of the art [ C. Meier, U. Muus, J. Renze, L. Naesens, E. De Clercq, J. Balzarini; Comparative study of bis (benzyl) phosphate triesters of 2 ', 3'-dideoxy-2', 3'-didehydrothymidine (d4T) and cycloSal-d4TMP hydrolysis, mechanistic insights and anti-HIV activity; Antiviral Chem. Chemother. 2002, 13, 101-114 ] to expect that the negative charge by a repulsive interaction with the enzyme leads to a slowing of the hydrolysis.

task It is the object of the present invention to provide improved nucleoside prodrugs, In particular, nucleoside analogs to provide those from the The prior art does not have known disadvantages.

Surprisingly manages to solve this problem by appropriate masking Nucleoside diphosphates and nucleoside triphosphates or their Analogs, where the masking only at the terminal phosphate is performed while on the masking of the internal phosphate is dispensed with. With the invention For the first time ever, the bioreversible masking of nucleoside diphosphates becomes possible (NDP) and nucleoside triphosphates (NTP) and their nucleoside analogues reached.

Detailed description the invention

oder der allgemeinen Formel (II)

oder ein pharmazeutisch annehmbares Salz davon,
wobei R¹, R⁴, R⁷ und R⁸ unabhängig voneinander H,

sind,
R Alkyl, Aryl oder OAlkyl ist,
R², R³, R⁵ und R⁶ unabhängig voneinander H, Alkyl, Aryl oder OAlkyl sind,
R⁹ ein Nukleosid, Nukleosidanalogon oder Alkoholrest ist, und
Kat⁺ ein Kation ist,
mit der Maßgabe, dass R¹, R⁴, R⁷ und R⁸ nicht alle H sind.The invention relates in a first aspect to a compound of general formula (I)

or the general formula (II)

or a pharmaceutically acceptable salt thereof,
where R ¹ , R ⁴ , R ⁷ and R ⁸ independently of one another H,

are,
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} a nucleoside, nucleoside analog or alcohol residue, and
Kat ^{+ is} a cation,
with the proviso that R ¹ , R ⁴ , R ⁷ and R ^{8 are} not all H.

Under a nucleoside is understood to mean organic molecules, consisting of a sugar residue and an organic base, in particular a nitrogen-containing heterocyclic base (nucleobase) exist. Of the Sugar residue is usually a pentose, z. B. 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 called nucleotides. The phosphate, diphosphate (pyrophosphate) or triphosphate group is usually with the 5'-C atom of the sugar component linked to the nucleoside.

Under a nucleoside analogue is understood here to be a nucleoside which naturally not in the human body occurs naturally in the human body However, occurring nucleoside but resembles that, for example processed by the cell according to the natural nucleoside, For example, phosphorylated and incorporated into a DNA strand becomes.

Of the Term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (eg, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl), branched chain Alkyl groups (e.g., isopropyl, tert -butyl, isobutyl), cycloalkyl (e.g. Alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, Cycloheptyl, cyclooctyl), alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups. Includes "alkyl" further alkyl groups, the oxygen, nitrogen, sulfur or Have phosphorus atom containing one or more carbon atoms of the Replace hydrocarbon skeleton. The term "alkyl" also includes both unsubstituted alkyls and substituted alkyls, wherein the latter relates to alkyl radicals having substituents, which is a hydrogen atom on one or more carbon atoms replace the hydrocarbon skeleton. Such 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), 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 can be further substituted, for. B. with the substituents indicated 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.

Be under "aryl" Groups understood with aromaticity, including 5- and 6-membered aromatic single ring groups containing zero to may include four heteroatoms, as well as multicyclic Systems with at least one aromatic ring. examples for Aryl groups include benzene, phenyl, pyrrole, furan, thiophene, Thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, Isooxazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. In addition, the term "aryl" includes multicyclic Aryl groups, e.g. B. tricyclic, bicyclic, z. Naphthalene, Benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, Methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, Benzofuran, purine, benzofuran, deazapurine or indolizine. Be under "aryl" Also understood aryl groups, the heteroatoms in the ring structure have ("heteroaryls"). The aromatic ring may be substituted at one or more ring positions. Aryl groups can also be used with alicyclic or heterocyclic rings, that are not aromatic, fused or bridged, so that a multicyclic system is formed (eg tetralin, Methylenedioxyphenyl).

Under an "alcohol residue" is here any organic Understood carbon compound in which a hydrogen atom on Carbon skeleton is replaced by a hydroxyl group. Examples of alcohols include sugars such as glucose, fructose, Mannose etc, but also other compounds such. B. geraniol.

The Compounds according to the invention can be used advantageously as prodrugs (prodrugs). They enable the introduction of nucleosides for the first time and nucleoside analogues as well as other organic compounds a hydroxyl group, e.g. As sugars or alcohols, on the Level of di- and triphosphates in the cell. This will for example possible for the first time, the nucleoside triphosphates used by the cell to insert directly into the cell, so that otherwise required no further intracellular phosphorylation steps more needed. This can not just be the actual use allows the introduced nucleosides through the cell better but also side effects, for example, by monophosphates are caused, are now avoidable. The invention Compounds can be used both as antiviral and as a Antitumor agent find advantageous use as a drug. They are especially useful as medicines for the treatment of infections Viruses, in particular retroviruses such as the HI virus. The connections But also suitable for analytical purposes in biochemical Investigations, as the phosphorylated metabolites intracellular be released. This will allow investigations that could not be done yet. Also Non-nucleosidic alcohols may be in the form of their di- or triphosphates transported into the cell and released there become.

With the help of the radicals R ² , R ³ , R ⁵ , R ⁶ , 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.

und R⁷ und R⁸ sind beide H.In a preferred embodiment, in the compound of the invention R ¹ and R ^{4 are} both

and R ⁷ and R ⁸ are both H.

und R¹ und R⁴ sind H.In an alternative preferred embodiment, in the compound of the invention R ⁷ and R ^{8 are} both

and R ¹ and R ⁴ are H.

More preferably, R ⁹ in the compound of the invention is a nucleoside or nucleoside analog. However, 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 of this invention are bis (4-acyloxybenzyl) -nucleoside diphosphate (BAB-NDP) and bis (4-acyloxybenzyl) -nucleoside triphosphate (BAB-NTP) and the corresponding ortho-isomers bis (2-acyloxybenzyl) -nucleoside diphosphate (BAB -NDP) and bis (2-acyloxybenzyl) -nucleoside triphosphate:

If the corresponding amide radical is used instead of an acyloxy radical in the compounds represented above, bis (4-acylamino benzyl) -nucleoside diphosphate, bis- (N-acylaminobenzyl) -nucleoside diphosphate, bis- (N-acylaminobenzyl) -nucleoside triphosphate and bis- (N-acylaminobenzyl) -nucleoside triphosphate:

about The acyl radical R can be the stability in different Media control and at the same time the polarity of the connection adjust, so that an effective passive transport over the cell membrane can be reached. On a masking of non-terminal Phosphates are dispensed with.

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-NDP prodrug according to the invention is shown below:

The The invention also relates in a further aspect to a pharmaceutical Composition which is a compound of the invention and a pharmaceutically acceptable carrier. pharmaceutical 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 which are used for an administration to mammals, including the People who are suitable. The term "carrier" in the Meaning of the present invention refers to any organic or inorganic, natural or synthetic substance, which combines with the active ingredient to simplify the application can be. Examples of such carriers include but are not limited to, organic or inorganic Solvent, 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. The term "pharmaceutical acceptable "means any mammal, In particular, humans, non-toxic material, the effectiveness the biological activity of the active ingredient substantially not impaired. Such materials can pharmaceutically acceptable levels of salts, buffers, preservatives, or the like. Non-limiting examples pharmaceutically acceptable carrier include magnesium carbonate, Magnesium stearate, talc, sugar, lactose, ethanol, glycerine, water etc. The pharmaceutical composition can be about also include auxiliary and / or diluents.

In In another aspect, the invention also relates to a pharmaceutical Dosage form which is a compound of the invention and a pharmaceutically acceptable carrier. Especially it is preferably a dosage form for oral Administration, for example a tablet or capsule.

oder der allgemeinen Formel (II)

oder eines pharmazeutisch annehmbaren Salzes davon,
wobei R¹, R⁴, R⁷ und R⁸ unabhängig voneinander H,

sind,
R Alkyl, Aryl oder OAlkyl ist,
R², R³, R⁵ und R⁶ unabhängig voneinander H, Alkyl, Aryl oder OAlkyl sind,
R⁹ ein Nukleosid, Nukleosidanalogon oder Alkoholrest ist, und
Kat⁺ ein Kation ist,
mit der Maßgabe, dass R¹, R⁴, R⁷ und R⁸ nicht alle H sind,
umfassend die Schritte des

• a) Inreaktionbringens eines Phenylesters der allgemeinen Formel (III)
  
  mit Dichlorophosphoramidit
  
• b) Inreaktionbringens des in Schritt a) erhaltenen Phosphoramidits der allgemeinen Formel (IV)
  
  mit einer Verbindung gemäß der allgemeinen Formel (V)
  
  oder mit einer Verbindung der allgemeinen Formel (VI)
  

In yet another aspect, the present invention relates to a process for the preparation of a compound of the general formula (I)

or the general formula (II)

or a pharmaceutically acceptable salt thereof,
where R ¹ , R ⁴ , R ⁷ and R ⁸ independently of one another H,

are,
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} a nucleoside, nucleoside analog or alcohol residue, and
Kat ^{+ is} a cation,
with the proviso 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)
  
  with dichlorophosphoramidite
  
• b) reacting the phosphoramidite of the general formula (IV) obtained in step a)
  
  with a compound according to the general formula (V)
  
  or with a compound of general formula (VI)
  

It The result is the corresponding diphosphate compounds (I) or triphosphate compounds (II).

sind und R⁷ und R⁹ werden so gewählt, dass beide H sind.In a preferred embodiment of the process according to the invention, R ¹ and R ⁴ are chosen such that both

and R ⁷ and R ⁹ are chosen so that both are H.

sind und R¹ und R⁴ werden so gewählt, dass beide H sind.In another preferred embodiment of the method according to the invention R ⁷ and R ⁸ are chosen so that both

and R ¹ and R ⁴ are chosen so that both are H.

In a particularly preferred embodiment of the method according to the invention, R ^{9 is} a nucleoside or nucleoside analog.

Alone for illustrative purposes, the invention will be described below of exemplary embodiments and the accompanying figures explained in more detail.

1 Chemical hydrolysis of BAB-AZTDP 185a to AB-AZTDP 186 (top after 12 h, bottom to 4 d); Snippets from the ³¹ P NMR spectra ( ¹ H decoupled). AcO = acyloxy.

2 Section from the ³¹ P NMR spectrum ( ¹ H decoupled) of the hydrolysis of 186 after 8 days to 222.

3 Comparison of hydrolysis half-lives.

4 Stability of the prodrugs of the invention in human blood plasma.

embodiments

In general, the synthesis of the compounds (I) and (II) according to the invention can be carried out using the example of nucleoside di- and nucleoside triphosphates (R9 = nucleoside) according to the following scheme:

phenylester III are reacted with dichlorophosphoramidite 194, preferably under inert gas (eg nitrogen), with cooling (eg -78 ° C) and in abs. Triethylamine (TEA), solved in abs. Tetrahydrofuran (THF). The resulting Phosphoramidite IV is then treated with a corresponding nucleoside monophosphate (NuclMP) or nucleoside diphosphate (NuclDP) to the corresponding Nucleoside di (Ia) or triphosphates (IIa) implemented.

Representation of BAB-NDP prodrugs

The basic representation of BAB-NDP prodrugs (181) according to the invention is illustrated below by way of example in the form of a retrosynthetic analysis of BAB-NDP prodrugs 181 (NuclMP = nucleoside monophosphate, Nucl = nucleoside):

The corresponding NTP prodrugs can be prepared instead of the nucleoside monophosphates (NuclMP) the corresponding Nucleoside diphosphates are used. The synthesis of all the invention Compounds I and II can according to the example BAB-NDP prodrugs described procedures.

The synthesis of the phenyl ester 193 by acylation is shown schematically below:

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 ° C. For 20 minutes, the respective acid chlorides (1.1 eq.), Dissolved in abs. THF, dripped. After 1-2 h reaction time at 0 ° C triethylammonium chloride is removed by filtration and the solvent is condensed off in vacuo. The residue is taken up in dichloromethane (DCM), washed twice with saturated sodium carbonate solution and once with H ₂ O, dried over sodium sulfate, filtered and the solvent removed in vacuo. The products are finally purified by either crystallization or chromatography.

Synthesis of dichlorophosphoramidite

Synthese der Phosphoramidite:

Phosphorus trichloride is reacted with diisopropylamine DIPA in diethyl ether. This is followed by Schlenk filtration. The result is dichlorophosphoramidite (194).

Synthesis of phosphoramidites:

The reactions are carried out under nitrogen as an inert gas. To 1.0 eq. Dichloro-N, N-diisopropylaminophosphoramidite, dissolved in abs. THF and cooled to -78 ° C, 2.2 eq. of the respective ester as well as 2.3 eq. Section. Triethylamine (TEA), dissolved in THF, added dropwise over 1 h. After complete addition, stirring is continued at RT for 16-20 h. Triethylammonium chloride is filtered off under inert gas, washed with THF and the filtrate is freed from the solvent in vacuo. It is u. U. necessary to repeat the filtration. The crude products are purified on chromatotron with PE and an ethyl acetate gradient. Synthesis of Nucleoside Monophosphates (NMP):

Variant A: The reaction is carried out under nitrogen as an inert gas. It will be 2.0 eq. POCl _{3 dissolved} in trimethyl phosphate and cooled to 0 ° C. This will be 1.0 eq. of the corresponding nucleoside and stirred at 0 ° C for a further 2 h. The reaction solution is then treated with saturated ammonium bicarbonate solution adjusted to pH = 8 and stirred for a further 10 min. The solution is frozen and freeze-dried. Finally, it is repeatedly chromatographed over an RP-18 phase with pure water.

Variant B: The reactions are carried out under nitrogen as an inert gas. It will be 4.4 eq. POCl 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), the reaction is terminated by addition of ice water and stirred for a further hour in the refrigerator. Careful addition of solid ammonium bicarbonate sets a pH of 8. 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.

Ion exchange on nucleoside monophosphates (NMP)

variant A: The NMPs are via a protonated ion exchange column Dowex 50WX8 elutes. The protonated form of the NMP is used with the each cationic hydroxide solution to the neutral point titrated or by direct addition of 2.0 eq. the respective Solutions neutralized and the solvent through Freeze-drying removed.

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, m / m) was added and the solvent removed by lyophilization.

Synthesis of exemplary bis (4-acyloxybenzyl) diphosphate prodrugs

Ammonium β-bis (4-acetoxybenzyl) d4TDP; (BAB-d4TDP)

The reaction is carried out under nitrogen as an inert gas. 0.16 g of bis (tetra-n-butylammonium) d4TMP (0.20 mmol, 1.0 eq.) And 94 mg of bis (4-acetoxybenzyl) N, N-diisopropylaminophosphoramidite (0.20 mmol, 1.0 eq.) In 3 mL abs. Acetonitrile dissolved. 36 mg of DCI (0.30 mmol, 1.5 eq.) Are added to the solution and the mixture is stirred at RT for 0.5 h. The solution is cooled to -25 ° C and oxidized with 51 μL t-BuOOH (5.5 M in n-decane, 0.28 mmol, 1.4 eq.). After 15 min at -25 ° 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 rechromatographed RP-18, first with water / methanol 2: 1, then 1: 1. The product is again freeze-dried. Yield: 14 mg (20 μmol, 10%) of a colorless, hygroscopic solid.
¹ H-NMR (400 MHz, MeOH) δ / ppm = 7.63 (q, 1H, J = 0.5 Hz, H _het -6), 7.40-7.34 (m, 4H, H-2), 7.08-7.02 (m, 4H, H-3), 6.94 (ddd, 1H, J = 1.5Hz, J = 1.5Hz, J = 3.3Hz, H-1 '), 6.38-6.34 (m, 1H, H-3'), 5.83 ( ddd, 1H, J = 2.0 Hz, J = 3.5 Hz, J = 6.0 Hz, H-2 '), 5.09 (dd, 4H, J = 6.2 Hz, J = 8.3 Hz, H-Bn), 4.96-4.92 ( m, 1H, H-4 '), 4.23-4.11 (m, 2H, H-5'), 2.27 (s, 6H, Ac), 1.89 (d, 3H, J = 1.0 Hz, Me _het ); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 171.1 (2 × C = O-Ac), 166.6 (C _het -4), 152.9 (C _het -2), 152.4 (2 × C-4), 138.7 (C _het -6), 135.3 (O-3 '), 134.9 (dd, J = 1.4 Hz, J = 7.1 Hz, 2 × C-1), 130.4 (d, J = 2.9 Hz, 4 × C-2), 127.6 (C-2 '), 123.0 (4 × C-3), 112.1 (C _het -5 ), 90.9 (C-1 '), 86.9 (d, J = 9.2 Hz, C-4'), 70.3 (dd, J = 1.7 Hz, J = 5.9 Hz, 2 × C-Bn), 68.1 (dd, J = 6.8 Hz, J = 6.8 Hz, C-5 '), 21.0 (2 × CH ₃ ), 12.5 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.20 (d, 1P, J = 19.6 Hz, P-□), -12.96 (d, 1P, J = 19.6 Hz, P- ☐); IR (KBr): □ [cm ^-1 ] = 3435, 2930, 1762, 1701, 1266, 1220, 1190, 1044, 1009, 912; UV (HPLC): λ _max = 265 nm; HPLC, method 1: t _R [min] = 9.41; _Rf (EE / MeOH 3: 2 v / v) = 0.59; MS (HR-ESI ^- ): calcd. 679.13 (M ^- ); found 679.11; C ₂₈ H ₃₃ N ₃ O ₁₄ P _2; Mol. Wt .: 697.52

¹H-NMR (400 MHz, DMSO-d₆)δ/ppm = 7.39-7.34 (m, 4H, H-2), 7.10-7.06 (m, 4H, H-3), 4.82 (d, 4H, J = 7.3 Hz, H-Bn), 2.26 (s, 6H, Ac); ¹³C-NMR (101 MHz, DMSO-d₆)δ/ppm = 169.2 (2 × C=O), 149.9 (2 × C-4), 135.6 (2 × C-1), 128.5 (4 × C-2), 121.6 (4 × C-3), 66.1 (d, J = 5.3 Hz, 2 × C-Bn), 20.9 (2 × CH₃); ³¹P-NMR (162 MHz, ¹H-decoupled, DMSO-d₆)δ/ppm = -1.42; ³¹P-NMR (162 MHz, DMSO-d₆)δ/ppm = –1.20-(–1.60) (m); C₁₈H₁₈O₈P Mol. Wt.: 393.31 Ammonium-β-bis-(4-acetoxybenzyl)AZTDP; (BAB-AZTDP)

It could be isolated by-product. This probably arises already during the coupling with incomplete drying of the nucleoside.

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ / ppm = 7.39-7.34 (m, 4H, H-2), 7.10-7.06 (m, 4H, H-3), 4.82 (d, 4H, J = 7.3 Hz, H-Bn), 2.26 (s, 6H, Ac); ¹³ C-NMR (101 MHz, DMSO-d ₆ ) δ / ppm = 169.2 (2 x C = O), 149.9 (2 x C-4), 135.6 (2 x C-1), 128.5 (4 x C). 2), 121.6 (4 × C-3), 66.1 (d, J = 5.3 Hz, 2 × C-Bn), 20.9 (2 × CH ₃ ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, DMSO-d ₆₎ δ / ppm = -1.42; ³¹ P-NMR (162 MHz, DMSO-d ₆ ) δ / ppm = -1.20 - (- 1.60) (m); C ₁₈ H ₁₈ O ₈ P Mol. Wt .: 393.31 Ammonium β-bis- (4-acetoxybenzyl) AZTDP; (BAB-AZTDP)

The reaction is carried out under nitrogen as an inert gas. 213 mg of bis (tetra-n-butylammonium) AZTMP (0.26 mmol, 1.0 eq.) In 3 ml abs. Acetonitrile dissolved: Now 0.16 g of bis (4-acetoxybenzyl) N, N-diisopropylaminophosphor-amidite (0.35 mmol, 1.4 eq.) And 44 mg DCl (0.38 mmol, 1.5 eq.) Was added and stirred at RT for 1.5 h. It is then cooled to -25 ° C and oxidized by adding 46 μL of t-BuOOH (5.5 M in n-decane, 0.25 mmol, 1.0 eq.). After 15 min at -25 ° 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. It will be one final purification on RP-18 reverse phase (water / methanol 1: 2). The product is freeze-dried.
Yield: 56 mg (76 μmol, 29%) of a colorless, hygroscopic solid.
¹ H-NMR (400 MHz, MeOH) δ / ppm = 7.71 (q, 1H, J = 1.2 Hz, H _het -6), 7.45-7.37 (m, 4H, H-2), 7.10-7.05 (m, 4H, H-3), 6.20 (dd, 1H, J = 6.3 Hz, J = 6.0 Hz, H-1 '), 5.18-5.08 (m, 4H, H-Bn), 4.40 (ddd, 1H, J = 3.5 Hz, J = 3.5 Hz, J = 7.1 Hz, H-3 '), 4.20 (ddd, 1H, J = 3.0 Hz, J = 4.9 Hz, J = 11.5 Hz, H-5'), 4.12 (ddd, 1H, J = 2.7Hz, J = 5.5Hz, J = 11.6Hz, H-5 '), 4.03 (ddd, 1H, J = 2.8Hz, J = 2.8Hz, J = 6.0Hz, H-4'), 2:40 to 2:23 (m, 2H, H-2 '), 2.27 (s, 6H, CH _3), 1.89 (d, 3H, J = 1.1 Hz, Me _het); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 171.0 (2 × C = O), 166.4 (C _het -4), 152.6 (2 × C-4), 152.5 (C _het -2), 137.8 ( C _het -6), 134.9 (dd, J = 1.9 Hz, J = 7.5 Hz, 2 x C-1), 130.5 (d, J = 2.0 Hz, 4 x C-2), 123.0 (4 x C-3 ), 112.2 (C _het -5), 85.8 (C-1 '), 84.4 (d, J = 9.0 Hz, C-4'), 70.4 (d, J = 5.6 Hz, 2 x C-Bn), 67.3 (d, J = 6.0 Hz, C-5 '), 62.5 (C-3'), 38.1 (C-2 '), 20.1 (2 x CH ₃ ), 12.7 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.17 (d, 1P, J = 21.1 Hz, P-□), -12.80 (d, 1P, J = 20.6 Hz, P- ☐); IR (KBr): □ [cm ^-1 ] = 3431, 3196, 3071, 1760, 1699, 1509, 1469, 1274, 1220, 1010, 967; UV (HPLC): λ _max = 265 nm; HPLC, method 1: t _R [min] = 10.32; mp = 90-92 ° C; R _f (H ₂ O / MeOH 1: 2 v / v, RP-18) = 0.63; MS (HR-ESI ^- ): calcd. 722.126 (M ^- ); found 722,126; C ₂₈ H ₃₄ N ₆ O ₁₄ P _2; Mol. Wt .: 740.55 ammonium β-bis (4-isobutyryloxybenzyl) AZTDP; (BIB AZTDP)

• Anmerkung: In der Überschrift ist von Ammonium als Gegenion die Rede. Das trifft nur teilweise zu, da das Produkt am Ende mit gemischten Gegenionen in etwa der Zusammensetzung NBu₄/NH₄ 1:3 erhalten wird. In dieser Form sind dann auch die NMR-Daten angegeben. Auf einen erneuten Ionenaustausch wird verzichtet, da bei einmaligem Ionenaustausch ca. 7% Zersetzungsprodukt in dem vormals reinen Produkt auftraten.

The reaction is carried out under nitrogen as an inert gas. 115 mg of bis (tetra-n-butylammonium) AZTMP (139 μmol, 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. Acetonitrile added. To this solution is added 109 mg of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidite (211 μmol, 1.5 eq.) And the reaction started by adding 26 mg of DCI (0.22 mmol, 1.6 eq.). After 1.5 h at RT, the reaction solution is cooled to -25 ° C and oxidized by adding 38 μL of t-BuOOH (5.5 M in n-decane, 0.21 mmol, 1.5 eq.). After 15 min at -25 ° C is warmed to RT and the solvent removed in vacuo. It is first by RP-18 chromatography with water / methanol 1: 1 remaining starting material and DCI removed from the crude product and then the product washed with pure methanol from the RP-18 phase. Parts of the methanol are removed on a rotary evaporator at low temperature, then with dist. Water filled and freeze-dried. The resulting 144 mg of crude product are passed through an ion exchanger (Dowex 50WX8, NH ₄ ⁺ , 10% acetonitrile is added to the water to dissolve the crude product). The eluate is lyophilized. With the obtained 102 mg of crude product, an RP-18 chromatography is again carried out, this time with methanol / water 3: 1. The product is finally lyophilized.
Yield: 53 mg (67 μmol, 48%) of a colorless solid.
¹ H-NMR (400 MHz, MeOD) δ / ppm = 7.72 (q, 1H, J = 1.2 Hz, H-6), 7.46-7.34 (m, 4H, H-2), 7.10-7.00 (m, 4H , H-3), 6.21 (dd, 1H, J = 6.2 Hz, J = 6.0 Hz, H-1 '), 5.18-5.10 (m, 4H, H-Bn), 4.40 (ddd, 1H, J = 3.2 Hz, J = 3.5 Hz, J = 6.4 Hz, H-3 '), 4.26-4.08 (m, 2H, H-5'), 4.04 (ddd, 1H, J = 2.8 Hz, J = 2.8 Hz, J = 5.8 Hz, H-4 '), 2.82 (sept., 2H, J = 7.0 Hz, CH-i-bu), 2.38-2.20 (m, 2H, H-2') 1.91 (d, 3H, J = 1.1 Hz, Me _het ), 1.30 (d, 12H, J = 7.0 Hz, CH ₃ -i-Bu); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 177.1 (2 × C = O), 165.0 (C _het -4), 152.6 (2 × C-4), 152.4 (C _het -2), 137.8 ( C _het -6), 134.9 (m, 2 x C-1), 130.5 (4 x C-2), 122.9 (4 x C-3), 112.2 (C _het -5), 85.8 (C-1 ') , 84.4 (d, J = 9.2 Hz, C-4 '), 70.4 (d, J = 5.6 Hz, 2 x C-Bn), 67.3 (d, J = 5.9 Hz, C-5'), 62.5 (C -3 '), 38.1 (C-2'), 35.3 (2 x CH-i-bu), 19.3 (4 x CH ₃ -i-bu), 12.7 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.16 (d, 1P, J = 21.1 Hz, P-α), -12.77 (d, 1P, J = 20.9 Hz, P- β); IR (KBr): □ [cm ^-1 ] = 3431, 3194, 3069, 2976, 2109, 1757, 1700, 1470, 1273, 1112, 1011; UV (HPLC): λ _max = 265 nm; HPLC, method 2: t _R [min] = 17.65; mp = 79 ° C; R _t (EE / MeOH 7: 3 v / v) = 0.47; R _f (H ₂ O / MeOH 1: 3 v / v, RP-18) = 0.64; MS (HR-ESI ^- ): calcd. 778.189 (M ^- ); found 778,195; C ₃₂ H ₄₂ N ₆ O ₁₄ P _2; Mol. Wt .: 796.66 ammonium β-bis- (4-pivaloyloxybenzyl) AZTDP; (BPB-AZTDP)

• Note: The title refers to ammonium as the counterion. This is only partially true, as the product is ultimately obtained with mixed counterions of about the composition NBu ₄ / NH ₄ 1: 3. In this form, the NMR data are then given. Renewed ion exchange is dispensed with, since with a single ion exchange about 7% decomposition product occurred in the formerly pure product.

variant A: The reaction is carried out under nitrogen as an inert gas. 146 mg of bis (tetra-n-butylammonium) AZTMP (176 μmol, 1.0 eq.) are initially 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 still twice with abs. Washed acetonitrile and the washing solution also transferred to the reaction flask. The Feedstock is still thrice with a few mL abs. Acetonitrile coevaporates, so that a colorless foam was created. This is in 2 mL abs. Acetonitrile added. To the solution are added 0.15 g of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidite (0.28 mmol, 1.6 eq.) And the reaction by adding started from 36 mg DCI (0.30 mmol, 1.7 eq.). After 1.75 h at RT, the reaction solution is a further 50 mg of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidit (88 μmol, 0.5 eq.) And 10 mg DCI (88 μmol, 0.5 eq.) And stirred for 2 h at RT. The reaction solution cooled to -25 ° C and by adding 64 μL of t-BuOOH (5.5 M in n-decane, 0.35 mmol, 2.0 eq.). After 15 min at -25 ° C is warmed to RT and the solvent in vacuo away. The crude product is enriched via an RP-18 phase. For this purpose, starting material and DCI with water / methanol Elutes 1: 1, then becomes isocratic successively first eluted with water / methanol 2: 1, 3: 1 and 5: 1. A big part of the product is mixed with the last eluent mixture from the column eluted (F2, 105 mg). However, the product is also already fairly high in the fractions eluting with water / methanol 2: 1 had been. Interestingly, these were the purer fractions (F1, 67 mg). F1 and F2 are separated by an ion exchanger converted into the ammonium salt. F1 will be again separated on an RP-18 phase, this time with methanol / water 2: 1, then 3: 1. It was possible to isolate 29 mg of the pure product and 20 mg of a mixed fraction. F2 is via a Sephadex G10 Phase eluted, but could not get clean in this way become. Also a separation on the preparative HPLC (acetonitrile / water 1.5: 1, RP-18, 10 mL / min) failed. renewed Chromatography on RP-18 silica gel with water / methanol 1.00: 2.25, then 1: 2.5, then 1: 3 did not deliver the pure product.

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.
Yield: 29 mg (35 .mu.mol, 20%) of a colorless solid, and significant amounts of product in mixed fractions.

Variant B: The reaction is carried out under nitrogen as an inert gas. There are 26 mg diammonium 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. To the solution are added 52 mg of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidite (95 μmol, 1.4 eq.) And 13 mg of DCI (0.11 mmol, 1.6 eq.) And stirred at RT for 1 h. A further 15 mg of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidite (27 μmol, 0.4 eq.) And 5 mg of DCI (42 μmol, 0.6 eq.) Are added and the mixture is stirred at RT for 3 h. The oxidation was carried out by adding 22 μL of t-BuOOH (5.5 M in n-decane, 0.12 mmol, 1.8 eq.) At -30 ° 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.
¹ H-NMR (400 MHz, MeOD) δ / ppm = 7.72 (q, 1H, J = 1.2 Hz, H-6), 7.45-7.36 (m, 4H, H-2), 7.06-7.00 (m, 4H , H-3), 6.21 (dd, 1H, J = 6.8 Hz, J = 6.8 Hz, H-1 '), 5.20-5.08 (m, 4H, H-Bn), 4.42-4.36 (m, 1H, H -3 '), 4.20 (ddd, 1H, J = 11.5 Hz, J = 3.0 Hz, J = 4.8 Hz, H-5'), 4.12 (ddd, 1H, J = 11.5 Hz, J = 2.7 Hz, J = 5.3 Hz, H-5 '), 4.04 (ddd, 1H, J = 2.7 Hz, J = 2.7 Hz, J = 5.6 Hz, H-4'), 3.26-3.18 (m, 3.2H, NBu ₄ ), 2.39 -2.23 (m, 2H, H-2 '), 1.91 (d, 3H, J = 1.1 Hz, Me _het ), 1.70-1.60 (m, 3.2H, NBu ₄ ), 1.46-1.36 (m, 3.2H, NBu ₄ ), 1.35 (s, 18H, t-Bu), 1.02 (t, 4.9H, J = 7.3 Hz, NBu ₄ ); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 178.5 (2 x C = O), 166.4 (C _het -4), 152.8 (2 x C-4), 152.4 (C _het -2), 137.8 ( C _het -6), 135.0-134.8 (m, 2 x C-1), 130.5 (d, J = 2.1Hz, 4 x C-2), 122.9 (4 x C-3), 112.2 (C _het -5 ), 85.8 (C-1 '), 84.4 (d, J = 9.1 Hz, C-4'), 70.4 (d, J = 5.7 Hz, 2 × C-Bn), 67.3 (d, J = 5.8 Hz, C-5 '), 62.5 (C-3'), 59.6-59.5 (m, NBu ₄ ), 40.1 (2 x C-tBu), 38.0 (C-2 '), 27.5 (6 x CH ₃ -t- Bu), 24.8, 20.8, 13.9 (NBu ₄ ), 12.7 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.18 (d, 1P, J = 20.3 Hz, P-α), -12.76 (d, 1P, J = 20.2 Hz, P- βαIR (KBr): □ [cm ^-1 ] = 3423, 3186, 2975, 2108, 1751, 1701, 1479, 1277, 1119, 1012, 968. UV (HPLC): λ _max = 265 nm; HPLC, Method 2: t _R [min] = 18.08, mp = 98-101 ° C, R _f (EE / MeOH 7: 3 v / v) = 0.70, MS (HR-ESI ^- ): calcd 806.221 (M ^- ); found 806,222; C ₃₄ H ₄₆ N ₆ O ₁₄ P ₂ ; Mol Wt .: 824.71 Ammonium β-bis (4-octanoyloxybenzyl) d4TDP; (BOB-d4TDP)

The reaction is carried out under nitrogen as an inert gas. 160 mg of bis (tetra-n-butylammonium) d4TMP (203 μmol, 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. Acetonitrile added. To the solution is added 0.22 g of bis (4-octanoyloxybenzyl) N, N-diisopropylaminophosphoramidite (0.35 mmol, 1.7 eq.) And the reaction started by adding 43 mg of DCI (0.37 mmol, 1.8 eq.). After 3 h at RT, the addition of 63 μL of t-BuOOH (5.5 M in n-decane, 0.35 mmol, 1.7 eq.) At -25 ° C is oxidized. After 15 minutes, the solvent is removed in vacuo, the residue taken up in water / acetonitrile and freeze-dried. The crude product is dissolved in a little methanol and chromatographed on a RP-18 phase, each isocratic with methanol / water 1: 1, 2: 1, 5: 1 and finally 6: 1. For the 1: 1 mixture remaining educt and DCI could be separated. The product fractions are combined and the methanol on a rotary evaporator at niedri The temperature is reduced to a few ml. It is filled with water and the solution is lyophilized. The resulting 150 mg are added via an ion exchanger (Dowex 50WX8, NH ₄ ⁺ , 30% acetonitrile in water) and the eluate is lyophilized. Another RP-18 chromatography with methanol / water 5: 1 gave the pure product. This is again freeze-dried. Yield: 114 mg (132 μmol, 65%) of a colorless solid.
¹ H-NMR (400 MHz, DMSO-d ₆₎ δ / ppm = 11.21 (s, 1H, NH), 7.65 (q, 1H, J = 1.2 Hz, H _het -6), 7:44 to 7:35 (m, 4H , H-2), 7.10-7.03 (m, 4H, H-3), 6.82 (ddd, 1H, J = 1.7 Hz, J = 1.7 Hz, J = 3.4 Hz, H-1 '), 6.34 (ddd, 1H, J = 1.6 Hz, J = 1.6 Hz, J = 6.0 Hz, H-3 '), 5.86 (ddd, 1H, J = 2.0 Hz, J = 3.1 Hz, J = 6.0 Hz, H-2'), 5.02 (dd, 4H, J = 3.8Hz, J = 6.5Hz, H-Bn), 4.88-4.84 (m, 1H, H-4 '), 4.10-3.88 (m, 2H, H-5'), 2.56 (t, 4H, J = 7.4 Hz, Hb), 1.78 (d, 3H, J = 1.0 Hz, Me _het ), 1.65 (tt, 4H, J = 7.3 Hz, J = 7.3 Hz, Hc), 1.40-1.20 (m, 16H, Hd, He, Hf, Hg), 0.87 (t, 6H, J = 6.8 Hz, Hh); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 173.8 (2 x Ca), 166.6 (C _het -4), 152.9 (C _het -2), 152.4 (2 x C-4), 138.7 (C _het -6), 135.3 (C-3 '), 134.9 (d, J = 7.0 Hz, 2 x C-1), 130.4 (d, J = 2.7 Hz, 4 x C-2), 127.6 (C-2' ), 122.9 (4 × C-3), 112.1 (C _het -5), 90.9 (C-1 '), 87.0 (d, J = 9.3 Hz, C-4'), 70.3 (dd, J = 1.8 Hz , J = 5.6 Hz, 2 × C-Bn), 68.2 (dd, J = 6.2 Hz, J = 6.2 Hz, C-5 '), 35.1 (2 × Cb), 32.9, 30.2, 30.1, 23.7 (2 × Cd, 2xCe, 2xCf, 2xCg), 26.0 (2xCc), 14.4 (2xCh), 12.5 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.11 (d, 1P, J = 20.6 Hz, P-□), -12.92 (d, 1P, J = 20.3 Hz, P ☐); IR (KBr): □ [cm ^-1 ] = 3188, 3071, 2956, 2927, 2856, 1756, 1701, 1467, 1263, 1112, 1010; UV (HPLC): λ _max = 264 nm; HPLC, method 2: t _R [min] = 21.52; mp = 73-75 ° C; R _f (EE / MeOH 7: 3 v / v) = 0.40; R _f (H ₂ O / MeOH 1: 5 v / v, RP-18) = 0.40; MS (HR-ESI ^- ): calcd. 847.297 (M ^- ); found 847,296; C ₄₀ H ₅₇ N ₃ O ₁₄ P ₂ ; Mol. Wt .: 865.84 ammonium β-bis (4-benzoyloxybenzyl) d4TDP; (BBB d4TDP)

The reaction is carried out under nitrogen as an inert gas. 178 mg of bis (tetra-n-butylammonium) d4TMP (226 .mu.mol, 1.0 eq.) Are first 2 h above 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 2.0 mL abs. Acetonitrile added. To this solution is added 216 mg of bis (4-benzoyloxybenzyl) N, N-diisopropylaminophosphoramidite (369 μmol, 1.6 eq.) And the reaction started by adding 46 mg of DCI (0.39 mmol, 1.7 eq.). After 1.5 h at RT, the reaction solution is cooled to -25 ° C and oxidized by the addition of 64 μL of t-BuOOH (5.5 M in n-decane, 0.35 mmol, 1.5 eq.). After 15 min at -25 ° C is warmed to RT and the solvent removed in vacuo. 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 passed through 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.
Yield: 62 mg (75 μmol, 33%) of a colorless solid.
¹ H-NMR (400 MHz, MeOH) δ / ppm = 8.19-8.13 (m, 4H, H-ortho), 7.70-7.64 (m, 3H, H-6, H-para), 7.56-7.50 (m, 4H, H-meta), 7.50-7.42 (m, 4H, H-2), 7.25-7.19 (m, 4H, H-3), 6.97 (ddd, 1H, J = 1.8Hz, J = 1.8Hz, J = 3.6Hz, H-1 '), 6.42 (ddd, 1H, J = 1.7Hz, J = 1.7Hz, J = 6.0Hz, H-3'), 5.86 (ddd, 1H, J = 2.2Hz, J = 3.6 Hz, J = 6.0 Hz, H-2 '), 5.16 (dd, 4H, J = 5.6 Hz, J = 8.5 Hz, H-Bn), 5.01-4.95 (m, 1H, H-4'), 4.28 -4.17 (m, 2H, H-5 '), 1.92 (d, 3H, J = 1.2 Hz, Me); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 166.6 (C _het -4), 166.5 (2 x C = O), 152.9 (C _het -2), 152.6 (2 × C-4), 138.8 (C _het -6), 135.4 (C-3 '), 135.2 (d, J = 7.0 Hz, 2 × C-1), 134.9 (2 × C-para), 131.1 (4 × C-ortho), 130.8 (2 × C-ipso), 130.6 (4 × C-2), 130.5 (4 × C-meta), 127.7 (C-2 '), 123.1 (4 × C-3), 112.1 (C _het -5), 90.9 (C-1 '), 87.0 (d, J = 9.2 Hz, C-4'), 70.3 (dd, J = 2.3 Hz, J = 5.6 Hz, 2 × C-Bn ), 68.2 (d, J = 6.2 Hz, C-5 '), 12.5 (Me); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.07 (d, 1P, J = 20.5 Hz, P-α), -12.86 (d, 1P, J = 20.5 Hz, P- β); IR (KBr): □ [cm ^-1 ] = 3435, 3199, 1734, 1692, 1452, 1268, 1202, 1063, 1025, 704; UV (HPLC): λ _max = 263 nm, 231 nm; HPLC, method 2: t _R [min] = 17.71; mp = 92 ° C; R _f (EE / MeOH 7: 3 v / v) = 0.38; MS (HR-ESI ^- ): calcd. 803.141 (M ^- ); found 803,139; C ₃₈ H ₃₇ N ₃ O ₁₄ P _2; Mol. Wt .: 821.66 ammonium β-bis (4-benzoyloxybenzyl) AZTDP; (BBB AZTDP)

The Reaction is performed as described for BBB-d4TDP. The following amounts are used: 96.0 mg of bis (tetra-n-butylammonium) AZTMP (116 μmol, 1.0 eq.), 108 mg of bis (4-benzoyloxybenzyl) N, N-diisopropylaminophosphor-amidite (184 μmol, 1.6 eq.) And 25 mg DCI (0.21 mmol, 1.8 eq.). Oxidized by addition of 36 μL t-BuOOH (5.5 M in n-decane, 0.20 mmol, 1.7 eq.).

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 re-chromatographed 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.
Yield: 58 mg (53 μmol, 46%) of a colorless solid as tetra-n-butylammonium salt; 3 mg (3 μmol, 3%) of a colorless solid as the ammonium salt. The NMR data are given for the ammonium salt.
¹ H-NMR (400 MHz, MeOH) δ / ppm = 8.18-8.13 (m, 4H, H-ortho), 7.74 (q, 1H, J = 0.5 Hz, H-6), 7.67 (tt, 2H, J = 7.3Hz, J = 2.5Hz, H-para), 7.57-7.45 (m, 8H, H-2, H-meta), 7.25-7.19 (m, 4H, H-3), 6.23 (dd, 1H, J = 6.8 Hz, J = 6.8 Hz, H-1 '), 5.25-5.10 (m, 4H, H-Bn), 4.46-4.40 (m, 1H, H-3'), 4.30-4.11 (m, 2H , H-5 '), 4.10-4.02 (m, 1H, H-4'), 2.44-2.26 (m, 2H, H-2 '), 1.91 (d, 3H, J = 0.9 Hz, Me _het ); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 166.5 (C _het -4), 166.4 (2 x C = O), 152.6 (C _het -2, 2 x C-4), 137.8 (C _het - 6), 135.1 (d, J = 5.6 Hz, 2 x C-1), 134.9 (2 x C-para), 131.1 (4 x C-ortho), 130.8 (2 x C-ipso), 130.6 (d, J = 1.6 Hz, 4 × C-2), 129.9 (4 × C-meta), 123.1 (4 × C-3), 112.2 (C _het -5), 85.9 (C-1 '), 84.4 (d, J = 9.1 Hz, C-4 '), 70.4 (d, J = 5.4 Hz, 2 × C-Bn), 67.3 (d, J = 5.8 Hz, C-5'), 62.5 (C-3 '), 38.1 (C-2 '), 12.7 (Me _het ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.18 (d, 1P, J = 19.4 Hz, P-α), -12.59 (d, 1P, J = 19.5 Hz, P- β); IR (KBr): □ [cm ^-1 ] = 3432, 3191, 3065, 2108, 1736, 1700, 1452, 1268, 1202, 1062, 1011, 707; UV (HPLC): λ _max = 261 nm, 231 nm; HPLC, method 2: t _R [min] = 17.79; mp = 98 ° C; R _f (EE / MeOH 7: 3 v / v) = 0.61; MS (HR-ESI ^- ): calcd. 846.158 (M ^- ); found 846,157; C ₃₈ H ₃₈ N 6 O ₁₄ P ₂ ; Mol. Wt .: 864.69 Ammonium β-bis- (4-benzoyloxybenzyl) 3'-O-acetyl-BVdUDP

The Reaction is carried out under nitrogen as an inert gas. The drying of the nucleotide and the reaction procedure carried out as described in the previous experiments.

It 50 mg of bis (tetra-n-butylammonium) 3'-O-acetyl-BVdUMP (53 μmol, 1.0 eq, in time for the reaction) with 48 mg of bis (4-benzoyloxybenzyl) N, N-diisopropylaminophosphoramidite (85 μmol, 1.6 eq.) And 12 mg DCI (102 μmol, 1.9 eq.) Implemented. Later will be again 10 mg amidite (0.4 eq.) And 4 mg DCI (0.5 eq.) added. The oxidation was carried out by adding 16 μL t-BuOOH (5.5 M in n-decane, 0.90 μmol, 1.7 eq.) at -25 ° C. The DC clearly showed product formation at. The solvent is removed in vacuo.

The purification of the crude mixture is carried out on a RP-18 phase: initially isocratic with water / methanol 1: 1, then 1: 3. The product fractions are combined and lyophilized. In the ³¹ P NMR spectrum, the product could be identified. It was still contaminated. After another reverse phase chromatography, the product could no longer be identified. Tetra-n-butylammonium-β-bis- (4-benzoyloxybenzyl) 3'-O-iso-butyryl-BVdUDP; (Ibu-BBB BVdUDP)

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. It will be 134 mg [NBu ₄ ] _1.66 [H] _0.34 -BVdUMP (weighted molecular weight about 926 g / mol, 145 μmol, 1.0 eq.) In 2 ml abs. Acetonitrile was combined with 136 mg of bis (4-benzoyloxybenzyl) N, N-diisopropylaminophosphoramidite (232 μmol, 1.6 eq.) And 30.0 mg of DCI (247 μmol, 1.7 eq.) At 0 ° C and then warmed to RT. After 40 minutes, another 6.0 mg of DCI (51 μmol, 0.4 eq.) And 26 mg of amidite (44 μmol, 0.3 eq.) Are added and stirred for 1 h. The oxidation was carried out by adding 50.0 μL t-BuOOH (5.5 M in n-decane, 276 μmol, 1.9 eq.) At -25 ° C. After 15 minutes, the solvent is 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 6: 1. Methanol is removed on a rotary evaporator, made up with water and lyophilized. The crude product is separated again via a RP-18 phase with methanol / water 5: 1. The product is finally lyophilized. Very good purification is achieved by additional chromatography using 0.25 M ammonium formate buffer instead of water.
Yield: 128 mg (104 μmol, 72%) of a colorless solid.
¹ H-NMR (400 MHz, MeOH) δ / ppm = 8.17-8.12 (m, 4H, H-ortho), 8.02 (s, 1H, H-6), 7.67 (tt, 2H, J = 7.0 Hz, J = 1.3Hz, H-para), 7.56-7.41 (m, 9H, H-2, H-meta, BVU-H-8), 7.25-7.17 (m, 4H, H-3), 7.05 (d, 1H , J = 13.5 Hz, BVU-H-7), 6.33 (dd, 1H, J = 5.7 Hz, J = 9.0 Hz, H-1 '), 5.40-5.36 (m, 1H, H-3'), 5.25 -5.15 (m, 4H, H-Bn), 4.32-4.22 (m, 2H, H-5 '), 4.22-4.17 (m, 1H, H-4'), 3.25-3.16 (m, 7H, Ha) , 2.59 (sept, 1H, J = 7.0 Hz, CH-ibu), 2.41-2.25 (m, 2H, H-2 '), 1.70-1.58 (m, 7H, Hb), 1.40 (tq, 7H, J = 7.5 Hz, J = 7.5 Hz, Hc), 1.16 (d, 6H, J = 7.1 Hz, CH ₃ -ibu), 1.00 (t, 11H, J = 7.4 Hz, Hd); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 177.8 (C = O-ibu), 166.4 (2 × C = O-Bz), 163.6 (C _het -4), 152.5 (2 × C-4) , 151.4 (C _het -2), 140.0 (C _het -6), 135.1 (dd, J = 3.2 Hz, J = 7.1 Hz, 2 x C-1), 134.9 (2 x C-para), 131.1 (4 × C-ortho), 130.8 (BVU-C-7), 130.7 (2 × O-ipso), 130.6 (d, J = 2.1 Hz, 4 × C-2), 129.8 (4 × C-meta), 123.0 (4 × C-3), 112.8 (C _het -5), 109.4 (BVU-C-8), 86.3 (C-1 '), 85.2 (d, J = 9.0 Hz, C-4'), 76.8 ( O-3 '), 70.4 (dd, J = 2.7 Hz, J = 5.7 Hz, 2 x C-Bn), 67.6 (d, J = 6.1 Hz, C-5'), 59.6-59.5 (m, 4 x Ca), 38.5 (C-2 '), 35.0 (CH-ibu), 24.8 (Cb), 20.8-20.7 (m, 4 x Cc), 19.2 (2 x CH ₃ -ibu), 14.0 (Cd); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -12.02 (d, 1P, J = 20.2 Hz, P-α), -12.64 (d, 1P, J = 20.4 Hz, P- β); IR (KBr): □ [cm ^-1 ] = 3434, 3063, 2964, 1876, 1736, 1712, 1682, 1465, 1267, 1200, 968; UV (HPLC): λ _max = 284 nm, 234 nm; HPLC, method 1: t _R [min] = 15.01; _Rf (EE / MeOH 4: 1 v / v) = 0.70; R _f (H ₂ O / MeOH 1: 6 v / v, RP-18) = 0.45; MS (HR-ESI ^- ): calcd. 981.104 (M ^- ); found 981,105; C ₅₉ H ₇₆ BrN ₃ O ₁₆ P ₂ ; Mol. Wt .: 1225.10 ammonium β-bis (4-benzoyloxybenzyl) carba-iso-ddADP; (BBB-carba-iso-ddADP)

• Anmerkung: Unter den Bedingungen an der präparativen HPLC wird das Gegenion ausgetauscht. Man erspart sich so einen Ionenaustausch über Dowex. Das Produkt konnte dennoch niemals vollständig rein isoliert werden. Wegen der geringen Mengen, die an der HPLC isoliert werden, ist das ¹³C-NMR für das Tetra-n-butylammoniumsalz angegeben. Die anderen Daten gelten für das Ammoniumsalz.
• Ausbeute: 36 mg (29 μmol, 25%, NBu₄-Salz) eines farblosen Feststoffes.
• ¹H-NMR (400 MHz, MeOD)δ/ppm = 8.48 (s, 1H, H_het-6), 8.21 (s, 1H, H_het-8), 8.17-8.09 (m, 4H, H-ortho), 7.70-7.63 (m, 2H, H-para), 7.57-7.40 (m, 8H, H-2, H-meta), 7.24-7.15 (m, 4H, H-3), 5.17 (d, 4H, J = 8.5 Hz, H-Bn), 4.83-4.73 (m, 1H, H-1'), 4.10-3.95 (m, 2H, H-6'), 2.50-2.27 (m, 2H, H-5', H-4'), 2.25-2.05 (m, 2H, H-2'), 2.04-1.76 (m, 3H, H-3', H-5'); ¹³C-NMR (101 MHz, MeOD)δ/ppm = 166.5 (2 × C=O-Bz), 161.6 (C_het-2), 152.6 (C_het-4), 151.9 (2 × C-4), 149.4 (C_het-6), 142.3 (C_het-8), 135.3 (d, J = 7.2 Hz, C-1), 134.9 (2 × C-para), 131.1 (4 × C-ortho), 130.8 (2 × C-ipso), 130.6 (4 × C-2), 129.8 (4 × C-meta), 128.6 (C_het-5), 123.0 (4 × C-3), 71.0 (d, J = 6.5 Hz, C-6'), 70.3 (d, J = 5.8 Hz, 2 × C-Bn), 59.7-59.5 (m, C-NBu₄), 57.1 (C-1'), 39.7 (d, J = 8.3 Hz, C-4'), 35.8 (C-5'), 32.1 (C-2'), 27.6 (C-3'), 24.8 (C-NBu₄), 20.8 (C-NBu₄), 14.0 (C-NBu₄); ³¹P-NMR (162 MHz, ¹H-decoupled, MeOD) δ/ppm = –11.18 (d, 1P, J = 21.8 Hz, P-☐), –13.00 (d, 1P, J = 20.9 Hz, P-☐); IR (KBr): ☐ [cm^–1] = 3423, 3210, 2955, 1733, 1617, 1510, 1426, 1268, 1202, 1063, 964, 700; UV (HPLC): λ_max = 305 nm, 225 nm; HPLC, Methode 4: t_R [min] = 12.53; mp = 102°C; R_f (EE/MeOH 7:3 v/v) = 0.22; MS (HR-ESI^–): calcd. 812.189 (M^–); found 812.184; C₃₉H₄₀N₆O₁₁P₂; Mol. Wt.: 830.72 Tetra-n-butylammonium-β-bis-(4-isobutyryloxybenzyl)PMEAMP
  

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. There are 85.0 mg [NBu ₄ ] _1.5 [H] _0.5 -BVdUMP (weighted molecular weight about 736 g / mol, 115 .mu.mol, 1.0 eq.) In 2 mL abs. Acetonitrile and 2 mL abs. DMF was combined with 109 mg of bis (4-benzoyloxybenzyl) N, N-diisopropylaminophosphoramidite (186 μmol, 1.6 eq.) And 24.0 mg of DCI (203 μmol, 1.8 eq.) At 0 ° C and then warmed to RT. After 40 minutes, a further 6.0 mg of DCI (51 μmol, 0.4 eq.) And 28 mg of amidite (48 μmol, 0.4 eq.) Are added and the mixture is stirred for 1 h. The oxidation was carried out by adding 42.0 μL of t-BuOOH (5.5 M in n-decane, 232 μmol, 2.0 eq.) At -25 ° C. After 15 minutes, the solvent is 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 on a RP-18 phase separated 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).

• Note: Under the conditions of the preparative HPLC, the counterion is exchanged. This saves an ion exchange via Dowex. Nevertheless, the product could never be completely isolated. Because of the small amounts that are isolated on HPLC, ¹³ C-NMR is reported for the tetra-n-butylammonium salt. The other data apply to the ammonium salt.
• Yield: 36 mg (29 μmol, 25%, NBu ₄ salt) of a colorless solid.
• ¹ H-NMR (400 MHz, MeOH) δ / ppm = 8.48 (s, 1H, H _het -6), 8.21 (s, 1H, H _het -8), 8.17-8.09 (m, 4H, H-ortho) , 7.70-7.63 (m, 2H, H-para), 7.57-7.40 (m, 8H, H-2, H-meta), 7.24-7.15 (m, 4H, H-3), 5.17 (d, 4H, J = 8.5 Hz, H-Bn), 4.83-4.73 (m, 1H, H-1 '), 4.10-3.95 (m, 2H, H-6'), 2.50-2.27 (m, 2H, H-5 ' , H-4 '), 2.25-2.05 (m, 2H, H-2'), 2.04-1.76 (m, 3H, H-3 ', H-5'); ¹³ C-NMR (101 MHz, MeOH) δ / ppm = 166.5 (2 × C = O-Bz), 161.6 (C _het -2), 152.6 (C _het -4), 151.9 (2 × C-4), 149.4 (C _het -6), 142.3 (C _het -8), 135.3 (d, J = 7.2 Hz, C-1), 134.9 (2 x C-para), 131.1 (4 x C-ortho), 130.8 ( 2 × C-ipso), 130.6 (4 × C-2), 129.8 (4 × C-meta), 128.6 (C _het- 5), 123.0 (4 × C-3), 71.0 (d, J = 6.5 Hz , C-6 '), 70.3 (d, J = 5.8 Hz, 2 x C-Bn), 59.7-59.5 (m, C-NBu ₄ ), 57.1 (C-1'), 39.7 (d, J = 8.3 Hz, C-4 '), 35.8 (C-5'), 32.1 (C-2 '), 27.6 (C-3'), 24.8 (C-NBu ₄ ), 20.8 (C-NBu ₄ ), 14.0 ( C-NBu ₄ ); ³¹ P-NMR (162 MHz, ¹ H-decoupled, MeOD) δ / ppm = -11.18 (d, 1P, J = 21.8 Hz, P-□), -13.00 (d, 1P, J = 20.9 Hz, P- ☐); IR (KBr): □ [cm ^-1 ] = 3423, 3210, 2955, 1733, 1617, 1510, 1426, 1268, 1202, 1063, 964, 700; UV (HPLC): λ _max = 305 nm, 225 nm; HPLC, method 4: t _R [min] = 12.53; mp = 102 ° C; R _f (EE / MeOH 7: 3 v / v) = 12:22; MS (HR-ESI ^- ): calcd. 812.189 (M ^- ); found 812,184; C ₃₉ H ₄₀ N ₆ O ₁₁ P ₂ ; Mol. Wt .: 830.72 Tetra-n-butylammonium-β-bis- (4-isobutyryloxybenzyl) PMEAMP
  

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. There are 0.29 g of [NBu ₄ ] ₂ PMEA (0.38 mmol, 1.0 eq.) In 4 mL abs. Acetonitrile and 3 mL abs. DMF was combined with 0.25 g of bis (4-isobutyryloxybenzyl) N, N-diisopropylaminophosphoramidite (0.49 mmol, 1.3 eq.) And 63 mg of DCI (0.53 mmol, 1.4 eq.) And then warmed to RT. After 45 min, it is oxidized at -20 ° C. with 89 μL t-BuOOH (5.5 M in n-decane, 0.49 mmol, 1.3 eq.). The solvents are removed in vacuo, the residue taken up in acetonitrile / water and lyophilized. The ³¹ P NMR spectrum contained no product signals.

Characterization of BAB-NDP prodrugs

• 1. Bestimmung der Hydrolysehalbwertszeiten t_1/2 bei verschiedenen pH-Werten (2.0, 6.8, 7.3 und 8.7) in unterschiedlichen Puffersystemen durch HPLC-Analytik.
• 2. Bestimmung der Hydrolyseprodukte, die bei der chemischen Hydrolyse entstanden, durch ³¹P-NMR-Spektroskopie und durch Auswertung der HPL-Chromatogramme unter Verwendung der synthetisierten Referenzverbindungen.
• 3. Bestimmung der Hydrolysehalbwertszeiten t_1/2 in verschiedenen biologischen Medien (CEM/0, Blutplasma, P3HR1, RPMI-Nährmedium, FCS).
• 4. Bestimmung der Hydrolyseprodukte, die bei der enzymatischen Hydrolyse entstanden, hauptsächlich durch Auswertung der HPL-Chromatogramme und in einem Fall durch Massenspektrometrie.

Generally, the procedure for each BAB NDP prodrug is as follows:

• 1. Determination of the hydrolysis _half- lives t _1/2 at different pH values (2.0, 6.8, 7.3 and 8.7) in different buffer systems by HPLC analysis.
• 2. Determination of the hydrolysis products resulting from the chemical hydrolysis by ³¹ P-NMR spectroscopy and by evaluation of the HPL chromatograms using the synthesized reference compounds.
• 3. Determination of the hydrolysis _half- life t _1/2 in various biological media (CEM / 0, blood plasma, P3HR1, RPMI culture medium, FCS).
• 4. Determination of the hydrolysis products formed during enzymatic hydrolysis, mainly by evaluation of the HPL chromatograms and in one case by mass spectrometry.

hydrolysis studies

pH-dependent hydrolysis kinetics

50 mM stock solutions of the target compounds in DMSO are prepared. 11 μL of these stock solutions are diluted in each case with 189 μL of deionized water and a further 100 μL of DMSO, so that 1.9 mM hydrolysis stock solutions are obtained. The hydrolysis is started by adding 300 μL of phosphate buffer solution (PBS, 50 mM, pH = 7.3, 37 ° C). The addition of internal standard is waived. The samples are incubated in a thermomixer at 37 ° C. Aliquots of the kinetics solution are then removed after different periods (60 μL each) and immediately frozen in liquid nitrogen. The samples are thawed individually and analyzed immediately. For this purpose, 40 μL are injected into the analytical RP-HPLC. To evaluate the kinetics, the chromatograms are evaluated at different UV wavelengths. The peak areas are plotted against the hydrolysis time. For evaluation, exponential compensation curves are placed through the individual measuring points with the aid of a spreadsheet program, so that the rate constants k of the hydrolyses could be determined. The hydrolysis half lives could now with the formula t 1.2 = In2 / k because the large excess of water was pseudo-first-order kinetics.

These Experiments are analogous for the determination of the hydrolysis half-lives at other pH values.

50 mM phosphate buffer (pH = 6.8, pH = 7.3, pH = 7.5, pH = 8.7 according to Sorensen):
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.

50 mM citrate-HCl buffer (pH = 2.0):
1.22 g of ammonium citrate are dissolved in 100 ml of deionized water and treated with conc. HCl adjusted to pH = 2.0.

Hydrolysis kinetics in cell extracts

It add 30 μL of the 50 mM stock DMSO solution of the compounds diluted with 220 μL DMSO to a concentration of 6.0 mM. In the following, 100 μL of cell extract (CEM / 0 for d4T, P3HR1 for BVdU and ACV, Mouse Liver Extract for different compounds) with 20 μL 70 mM magnesium chloride solution and add 20 μL of the 6.0 mM stock solution Pipette. For every reading becomes such a solution prepared and incubated at 37 ° C in a thermomixer. After the corresponding periods selected for a measurement The hydrolyses are made by adding 300 μL of methanol stopped and stored for 5 min at 0 ° C. Subsequently followed centrifugation for 10 min at 13000 rpm. The supernatant is taken up in syringes and filtered (Schleicher & Schuell Spartan 13/30, 0.2 μm) and frozen in liquid nitrogen (-196 ° C). The samples are at the analytical RP-HPLC cleaved into its components (injection: 90 μL). The evaluation was carried out without normalization of the peak areas.

Hydrolysis kinetics in human blood plasma

The hydrolysis kinetics in human blood plasma were analogous to the kinetics in cell extracts. In contrast, however, no MgCl ₂ solution is added. Diluted blood serum (20% or 50% in 50 mM PBS pH 6.8, given in the individual experiments) is added to the samples instead of the cell extract.

Hydrolysis kinetics in culture medium

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 is culture medium (RPMI) with 10% Heat Deactivated Fetal Calf Serum (FCS) added.

Chemical hydrolysis using the example of the invention BAB-NDP prodrugs BAB-AZTDP 185a.

AZT represents the nucleoside analogue 3'-azido-2 ', 3'-dideoxythymidine, also known as Retrovir.

The NMR spectroscopic monitoring of the hydrolysis (s. 1 ) 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. Starting from 186, only AZTDP 222 occurs as the hydrolysis product (s. 2 ).

This hydrolysis proceeds selectively. Only AZTDP 222 is released ( 2 ). Since it proceeds more slowly than the first hydrolysis, the intermediate can also be well detected. These results indicate that the chemical hydrolysis of BAB-AZTDP 185a occurs almost exclusively via the intermediate AB-AZTDP 186 with final quantitative release of the nucleotide AZTDP 222.

Comparison between chemical and enzymatic hydrolysis

By HPLC analysis showed the hydrolysis half lives of the compounds certainly.

The results of the hydrolysis studies in phosphate buffer or cell extracts are in 3 summarized.

Out 3 the big difference between chemical and enzymatic hydrolysis becomes clear. Also, one recognizes the increase in stability by branching the ester modification. Furthermore, the chemical stability increases when a mask has already been split off.

Of additional interest is the stability of the compounds in human blood plasma in order to assess whether transport of the intact prodrug to the site of action can take place. Although the tested compounds lost stability compared to the pH-dependent hydrolysis, they nevertheless had to be detected in incubation studies over several hours, such as 4 clarified. Thus a systemic distribution should be possible.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• DD Richman, MA Fischl, MH Grieco, MS Gottlieb, PA Volberding, OL Laskin, JM Leedom, J. Groopman, D. Mildvan, MS Hirsch, GG Jackson, DT Durack, D. Phil, S. Nusinoff-Lehrman; The toxicity of AZT in the treatment of patients with AIDS and AIDS-related complex; New Engl. J. Med. 1987, 317, 192-197. [0003]
• J. Balzarini, P. Herdewijn, E. De Clercq; Differential Patterns of Intracellular Metabolism of 2 ', 3'-Didehydro-2', 3'-Dideoxythymidine and 3'-Azido-2 ', 3'-Dideoxythymidine, Two Potent Anti-human Immunodeficiency Virus Compounds; J. Biol. Chem. 1989, 264, 6127-6133 [0005]
• - RJ Sawchuk, Z. Yang; Investigation of distribution, transport and uptake of anti-HIV drugs to the central nervous system; Advanced Drug Delivery Reviews 1999, 39, 5-31 [0006]
• A. Pompon, I. Lefebvre, J.-L. Imbach, S. Khan, D. Farquhar; Decomposition Pathways of the Mono- (Pivaloyloxymethyl) and Bis- (Pivaloyloxymethyl) Esters of Azidothymidine 5'-Monophosphates in Cell Extract and in Tissue Culture Medium - An Application of the Online Isrp-Cleaning HPLC Technique; Antiviral Chem. Chemother. 1994, 5, 91-98 [0008]
• I. Lefebvre, C. Perigaud, A. Pompon, A.-M. Aubertin, J.-L. Girardet, A. Kim, G. Gosselin, J.-L. Imbach; Mono Nucleosides Phosphotriester Derivatives with S-Acyl-2-thioethyl Bioreversible Phosphate Protecting Groups: Intracellular Delivery of 3'-azido-2 ', 3'-dideoxythymidine-5'-monophosphates; J. Med. Chem. 1995, 38, 3941-3950 [0008]
• - W. Thomson, D. Nicholls, WJ Irwin, JS Al-Mushadani, S. Freeman, A. Karpas, J. Petrik, N. Mahmood, AJ Hay, Synthesis, Bioactivation and Anti-HIV Activity of the Bis (4). acyloxybenzyl) and mono (4-acyloxybenzyl) esters of the 5'-monophosphate of AZT, J. Chem. Soc., Perkin Trans., 1993, 1, 1239-1245 [0008]
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• - W. Thomson, D. Nicholls, WJ Irwin, JS Al-Mushadani, S. Freeman, A. Karpas, J. Petrik, N. Mahmood, AJ Hay, Synthesis, Bioactivation and Anti-HIV Activity of the Bis (4). acyloxybenzyl) and mono (4-acyloxybenzyl) esters of the 5'-monophosphate of AZT, J. Chem. Soc., Perkin Trans., 1993, 1, 1239-1245 [0009]
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Claims (13)

Compound of the general formula (I)

or the general formula (II)

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ⁴ , R ⁷ and R ^{8 are} independently H,

R, R is alkyl, aryl or Oalkyl, R ² , R ³ , R ⁵ and R ^{6 are} independently H, alkyl, aryl or Oalkyl, R ^{9 is} a nucleoside, nucleoside analog or alcohol residue, and Kat ^{+ is} a cation with the proviso that R ¹ , R ⁴ , R ⁷ and R ^{8 are} not all H. Compound according to claim 1, characterized in that R ¹ and R ^{4 are} both

or

and R ⁷ and R ^{8 are} H. Compound according to claim 1, characterized in that A ⁷ and A ⁸ both

or

and R ¹ and R ^{4 are} H. Compound according to one of the preceding claims, characterized in that R ^{9 is} a nucleoside or nucleoside analogue. A compound according to any one of claims 1 to 4 for use as a medicine. A compound according to any one of claims 1 to 4 for use as an antiviral drug, preferably as a drug for the treatment of HIV infection. Use of a compound according to any one of the claims 1 to 4 for the preparation of a medicament, preferably an antiviral Drug, particularly preferably a drug for treatment an HIV infection. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 and a pharmaceutical acceptable vehicle. Pharmaceutical dosage form, in particular oral Dosage form comprising a compound according to one of the claims 1 to 4 and a pharmaceutically acceptable carrier. Process for the preparation of a compound of general formula (I)

or the general formula (II)

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ⁴ , R ⁷ and R ^{8 are} independently H,

R, R is alkyl, aryl or Oalkyl, R ² , R ³ , R ⁵ and R ^{6 are} independently H, alkyl, aryl or Oalkyl, R ^{9 is} a nucleoside, nucleoside analog or alcohol residue, and Kat ^{+ is} a cation with the proviso 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)

with dichlorophosphoramidite

b) reacting the phosphoramidite of the general formula (IV) obtained in step a)

with a compound according to the general formula (V)

or with a compound of general formula (VI)

A method according to claim 10, characterized in that R ¹ and R ^{4 are} both

or

and R ⁷ and R ^{8 are} H. A method according to claim 10, characterized in that R ⁷ and R ^{8 are} both

or

and R ¹ and R ^{4 are} H. Method according to one of claims 10 to 12, characterized in that R ^{9 is} a nucleoside or Nucleoside analogue.