DE19618727C2 - Preparation of alkylated nucleoside 3'-phosphates - Google Patents

Description

The invention relates to a process for the preparation of alkylated nucleoside-3'- Phosphates.

In the treatment of tumors, tumor therapeutics are often used, the effect of which is based on the alkylation of DNA. The effect of these tumor therapeutics is, however, affected by cellular repair mechanisms, e.g. B. the O ⁶ - alkyl guanosine transferase, reduced or even prevented. In order to counteract this, it could be useful to use alkylated nucleoside-3'-phosphates in addition to the tumor therapeutics mentioned. Alkylated nucleoside-3'-phosphates represent the substrate for the O ⁶ -alkyl guanosine transferase, whereby it would be bound in the tumors and prevented from their DNA repair activity. However, the production of alkylated nucleoside 3'-phosphates turns out to be extremely difficult.

The present invention is therefore based on the object of a method provide with which alkylated nucleoside 3'-phosphates can be produced.

According to the invention, this is achieved by the subject matter in the claims.

The present invention thus relates to a process for the preparation of an alkylated nucleoside 3'-phosphate, the nucleoside 3'-phosphate having a nucleobase, a pentose and a phosphate group, comprising the following steps:

• a) Implementation of a nucleoside-3'-phosphate having protecting groups with a diazoalkane as the alkylating agent and
• b) splitting off the protective groups,

the protecting groups on the pentose of the nucleoside 3'-phosphate Di methoxytrityl, silyl and / or acyl group, the protecting groups on the Phosphate group of the nucleoside 3'-phosphate are cyanoethyl groups and the protecting groups on the nucleobase of the nucleoside 3'-phosphate acyl are groups.  

The term "nucleobases" encompasses any basic heterocycle, e.g. B. Purine, pyrimidine or derivatives thereof, such as adenine, guanine, cytosine, uracil or Thymidine.

The term "pentose" includes any saccharide that has 5 carbon atoms, such as D-ribofuranose and 2-deoxy-D-ribofuranose. The pentose can over its carbon atom 1 must be β-glycosidically bound to a nitrogen atom of the nucleobase.

The term "phosphate group" includes organic and inorganic phosphates of any kind, e.g. B. mono-, di- or triphosphates. The phosphate group can be attached to the Carbon atom 3 and possibly 5 of the pentose may be bound.

Preferred nucleoside 3'-phosphates are adenosine, guanosine, cytidine, uridine, 2'- Deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytosine and thymidine-3'-phosphate, their monophosphates are very particularly preferred.

In the process according to the invention, protective groups are used in step (a) Nucleoside 3'-phosphates reacted with a diazoalkane as an alkylating agent. The nucleoside 3'- Phosphate can be the protective groups on OH groups of pentose, on OH groups of Have phosphate group (s) and possibly on the nucleobase. It is beneficial if Protect all OH groups of the pentose and the phosphate group (s) are. Protecting groups present on OH groups of the pentose according to the invention are dimetoxytrityl, Silyl and / or acyl groups such as phenylacetyl and acetyl groups. According to the present Protecting groups on OH groups of the phosphate group (s) are cyanoethyl groups. Protective groups on the nucleobase according to the invention are the above Acyl groups. The protecting groups on the nucleobase can be attached to any suitable Position of the nucleobase that is not to be alkylated, e.g. B. on one Amino group.

The preparation of nucleoside-3'-phosphates containing protective groups and the materials required for this purpose are known to the person skilled in the art. For example, these can be obtained from commercially available precursors (cf. implementation of 7 to 8 in FIG. 2). They can also be produced from the nucleosides (pentose and nucleobase) themselves (cf. FIG. 1).

As alkylating agents in step (a) of the process according to the invention Used diazoalkanes, such as diazomethane, Diazoethane and / or diazo-n-butane.

In step (a), several alkyl groups can also be introduced by the alkylation be, e.g. B. in different positions. The alkyl groups can be the same or be different from each other.

Step (a) produces alkylated, protecting group-containing nucleoside 3'-phosphates receive.

In step (b) of the process according to the invention, the protective groups are split off. This is advantageously carried out under mild conditions, so that the alkyl groups introduced in step (a) are not influenced. The elimination of dimethoxytrityl groups can, for. B. by means of an ion exchanger in the H ⁺ form. The silyl groups can be split off with fluoride. Acyl groups can be treated with bases, e.g. As ammonia or OH ^{- are} split off. Cyanoethyl groups or their derivatives can be split off by incubation with ammonia. The protective groups can be split off in succession or in a "one-pot reaction".

In a preferred embodiment of the inventive method can and / or after step (b) there is a cleaning and / or separation step. Is z. B. in Step (a) an isomer mixture of alkylated, protecting group-containing nucleoside Obtained 3'-phosphates, it is convenient to separate this into the isomers before the Protective groups are split off. The cleaning and / or separation step can e.g. B. with chromatographic methods, such as preparative HPLC.

Examples of the method according to the invention are shown in FIGS. 1 and 2.

The process according to the invention is characterized in that alkylated nucleoside 3'- Phosphates can be obtained in large quantities and high purity. Furthermore, that is Procedure easy to perform and inexpensive.

Alkylated nucleoside-3'- Phosphates comprising a nucleobase, a pentose and a phosphate group as they are are defined above, wherein there is an alkyl group on the nucleobase, available.

Alkylated nucleoside 3'-phosphates can be used in the treatment of tumors Tumor therapeutics, e.g. B. alkylating the DNA can be used. So that can be achieved become that these tumor therapeutics develop their full effect. Can also alkylated nucleoside 3'-phosphates can be used alone for the treatment of tumors.

Furthermore, alkylated nucleoside 3'-phosphates can be used for the analysis of DNA-alkyl adducts, e.g. B. in the ³² P post-labeling process. Alkylating compounds can cause cancer by alkylating bases of cellular DNA, creating DNA-alkyl adducts. Such adducts can be detected using the ³² P postlabeling method. For this purpose, the above DNA is degraded enzymatically to nucleoside 3'-phosphates, whereby a mixture of conventional nucleoside 3'-phosphates and alkylated nucleoside 3'-phosphates is formed. A radioactive phosphate group is then introduced enzymatically at the 5 'position using γ- ³² P-ATP. It is necessary for the nucleosides to have a phosphate group at the 3 'position, since only these can be labeled in the above manner. The mixture of the radioactively labeled nucleosides is then separated in a conventional manner and the radioactively labeled nucleosides are detected. With this method, DNA-alkyl adducts can be detected in very small amounts (approx. 1 adduct per 10 ⁹ normal nucleosides). Because of this small amount, structural proof of the DNA - alkyl adducts is not possible. In order to prove the structure of a DNA adduct, it is necessary to provide substances that can be used as reference substances in the ^32- postlabeling procedure. Such substances are alkylated nucleoside 3'-phosphates.

In addition to the ³² P postlabeling method, DNA-alkyl adducts are also detected with monoclonal antibodies. For this, the procedure is as described above, the alkylated nucleoside 3'-phosphates being detected by means of monoclonal antibodies directed against them. Alkylated nucleoside 3'-phosphates can be used to obtain the monoclonal antibodies.

Brief description of the drawings

Fig. 1 shows the preparation of O ⁶ -n-butyl-2'-deoxyguanosine-3'-monophosphate, and

Fig. 2 shows the preparation of 3 isomeric alkylated 2'-deoxythymidine-3'-monophosphates.

Compound 8 is prepared from commercially available compound 7 in a two-step one-pot reaction (1. tetrazole-catalyzed coupling with hydroxypropiononitrile; 2. oxidation with cumene hydroperoxide). Compound 8 is then directly with any diazoalkane, e.g. B. diazomethane, diazoethane or diazo-n-butane. In this reaction, the 3 isomeric compounds 9a-c are formed simultaneously. The compounds 9a-c are separated by means of preparative HPLC. The protecting groups are split off for each isomer separately. The dimethoxytrityl groups are split off in the H ⁺ form with an ion exchanger. The cyanoethyl groups are split off by incubation with ammonia. The alkylated nucleoside 3'-monophosphates 10a-c are obtained and are purified by preparative HPLC.

The following example illustrates the invention.

Example: Production of O ⁶ -n-butyl-2'-deoxyguanosine-3'-monophosphate

The structure and preparation of O ⁶ -n-butyl-deoxyguanosine-3'-monophosphate is shown in FIG. 1.

(a) Preparation of N ² -phenylacetyl-2'-deoxyguanosine 2 according to Synthesis, 1986 (1), 45-46 Solution 1

10 mmol (2.67 g) 2'-deoxyguanosine 1 was dissolved in 2 × 30 ml pyridine concentrated and suspended in 50 ml of pyridine. 50 mmol (6.5 ml) Trimethylchlorosilane slowly added dropwise. After 30 minutes it was Mix cooled with ice.

Solution 2

16.0 mmol (2.7 g) of hydroxybenzotriazole were treated with acetonitrile (4 × 30 ml) and concentrated in 5 ml of acetonitrile. Under air and Exclusion of moisture was added dropwise 15 mmol (2 ml) phenylacetic acid chloride. After 30 minutes, enough pyridine was added to dissolve the precipitate (about 0.3 ml).

Solution 2 was then placed in the cooled solution 1 under air and Exclusion of moisture dripped. After the addition had ended, the mixture was left overnight (15 Hours) warmed to room temperature. Then the reaction mixture chilled with ice. Then 10 ml of water and after 5 minutes 20 ml concentrated ammonia added. After a further 15 minutes it was in vacuo evaporated to dryness (water bath temperature: 30 ° C). The residue obtained was taken up in as much water (approx. 20 ml) as was necessary to dissolve. Then it was shaken with 50 ml of diethyl ether and the organic phase decanted. The aqueous phase was crystallized at 4 ° C. The crystallized Precipitate was filtered off and dried in vacuo. 3.0 g (78 %) 2 received.  

(b) Preparation of 5'-O- (4,4-dimethoxytrityl) -N ² -phenylacetyl-2'-deoxyguanosine 3 according to Nucleic Acid Research 1987 (15/2) 397-416)

5.7 mmol (2.18 g) 2 were concentrated with pyridine (3 × 10 ml), in pyridine (30 ml) suspended and cooled to 0 ° C. 6.3 mmol (2.13 g) dimethoxytrityl chloride added. It was overnight (approx. 15 Hours) stirred. Then 4 ml of methanol was added. After 15 minutes was concentrated in vacuo (water temperature 30 ° C). The backlog was in Dichloromethane (100 ml) added and with 5% aqueous Washed sodium carbonate solution (3 x 75 ml) and water (1 x 75 ml). The organic phase was decanted, dried over sodium sulfate and in vacuo constricted. The crude product was on silica gel with chloroform / ethanol = 95/5 chromatographed. 2.9 g (74%) 3 were obtained.

(c) Preparation of 5'-O- (4,4-dimethoxytrityl) -N ² -phenylacetyl-2'-deoxyguanosine-3'- bis (cyanoethyl) phosphate 4

1 mmol (0.68 g) 3 was concentrated with THF (2 × 10 ml) and in THF (10 ml) solved. 4 equivalents of Hünig base and then 2 Equivalents of cyanoethyl (bis-isopropyl) amido chlorophosphite were added dropwise and at Room temperature stirred. After 1 hour, 30 ml was added to the reaction mixture Ethyl acetate and 1.5 ml of triethylamine were added. The organic solution was with 10% sodium carbonate solution (2 × 10 ml) and saturated sodium chloride Solution (10 ml) washed, dried over sodium sulfate and under vacuum constricted. The crude product was cleaned using Silica gel column chromatography; Dichloromethane / ethyl acetate / triethylamine / methanol = 45/45/10 / 2.5. 0.6 g (67%) of an intermediate product was obtained.

0.11 mmol (100 mg) of this intermediate were dissolved in about 10 ml of acetonitrile dissolved, with 0.25 mmol (18 mg) hydroxypropiononitrile and with 0.5 mmol (35 mg) tetrazole added. After 1 hour, 1 equivalent became 3% Cumene hydroperoxide solution added in acetonitrile and at room temperature  stirred (TLC control, silica gel chlorofrom / methanol = 9/1). After about 1 hour a few drops of ethanol were added and the mixture was concentrated under vacuum. The Residue was taken up in chloroform (30 ml) and with 2% Washed sodium bicarbonate solution. The organic phase was over Dried sodium sulfate and concentrated in vacuo. The cleaning was done via column chromatography on silica gel with chloroform / methanol = 95/5). It 300 mg (61%) 4 were obtained.

(d) Preparation of O ⁶ -n-butyl-5'-O- (4,4-dimethoxytrityl) N ² phenylacetyl-2'-deoxyguanosine-3'-bis (cyanoethyl) phosphate 5

115 µmol (100 mg) 4 were dissolved in 5 ml of methanol and ethereal with 3 ml Diazo-n-butane solution added. After 30 minutes, the mixture was concentrated under vacuum and then chromated on silica gel (chloroform / ethanol = 98/2). 40 mg (37%) 5 were obtained.

(e) Preparation of O ⁶ -n-butyl-2-'deoxyguanosine-3'-phosphate 6

32 µmol (30 mg) of the fully protected 5 was dissolved in 3 ml of nitromethane and mixed with a solution saturated with zinc (II) bromide in 3 ml of nitromethane. After 15 minutes, the mixture was diluted with 50 ml of dichloromethane and the organic Phase shaken with 30 ml of 1 M ammonium acetate solution. The organic The phase was washed with water and saturated sodium chloride solution (10 ml each) and concentrated under vacuum. The crude product was dissolved in 5 ml of water and with concentrated aqueous ammonia (10 ml) for 15 hours at room temperature grows. It was then lyophilized, the residue in a little water added, shaken with 3 × 10 ml of chloroform and the aqueous solution lyophilized again.

4 mg of 6 were obtained.

Spectroscopic data from 6

ESI: -Q1MS LMR UP LR; Infusion 5 µl / min, MeOH
M (C ₁₄

H ₂₂

N ₅

O ₇

P) = 403; [MH] 402.0 (100%)
¹

H-NMR (D ₂

0.250 Mhz) δ = 8.08 (s, 1H, NH); 6.37 (dd, 1H, 1'-H); 4.92 (m, 1H, 3'-H); 4.48 (m, 2H, O ⁸

-CH ₂

); 4.33 (m, 1H, 4'-H); 3.84 (m, 2H, 5'-H); 2.70 (m, 2H, 2'-H ₂

) _;

1.80 (m, 2H, O ⁸

- C-CH ₂

) _;

1.50 (m, 2H, O ⁸

-CC-CH ₂

); 0.95 (t, 3H, O ⁸

-CCC-CH ₃

)

Claims (6)

1. A method for producing an alkylated nucleoside 3'-phosphate, the nucleoside 3'-phosphate having a nucleobase, a pentose and a phosphate group, comprising the following steps:

• a) reacting a protecting group-containing nucleoside 3'-phosphate with a diazoalkane as alkylating agent and
• b) splitting off the protective groups,

where the protecting groups on the pentose of the nucleoside 3'-phosphate are dimethoxytrityl, silyl and / or acyl group, the protecting groups on the phosphate group of the nucleoside 3'-phosphate are cyanoethyl groups and the protecting groups on the nucleobase of the nucleoside 3'-phosphate acyl groups. 2. The method according to claim 1, characterized in that the nucleobase has a purine or pyrimidine residue. 3. The method according to claim 1 or 2, characterized in that the pentose Is D-ribofuranose or 2-deoxy-D-ribofuranose. 4. The method according to any one of claims 1-3, characterized in that the Phosphate group is a mono-, di- or triphosphate. 5. The method according to any one of claims 1-4, characterized in that the protective groups on OH groups of pentose, on OH groups of phosph at group and / or on the nucleobase. 6. The method according to any one of claims 1-5, characterized in that before and / or after step (b) a cleaning and / or separation step is carried out becomes.