Phosphonate derivatives of acyclovir and the method of production thereof

 

(57) Abstract:

The invention relates to new biologically active phosphonate derivative of acyclovir. Describes phosphonate derivatives of 9-[(2-hydroxyethoxy)methyl]guanine (ACV) General formula:

where R' R'' represent alkyl, aryl or aralkyl. Also described is a method of obtaining phosphonate derivatives of acyclovir. The technical result obtained new compounds with selective Antiherpes virus effect of activity with efficiency exceeding the efficiency of ACV, and acting on the strains of the herpes simplex virus that are resistant to the action ACV, in addition, the claimed compounds can enter the effective therapeutic dose of the active substance in the pharmaceutical composition for use as drugs having Antiherpes virus effect of antiviral activity. 2 AD. and 2 C.p. f-crystals, 3 tables.

The invention relates to new biologically active phosphonate derivatives of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir, ACV), method of their production and pharmaceutical compositions based on these compounds, which can find application in medicine.

It is known that inhibition of reproduction of HSV can be at different stages of its life cycle, but it is obvious that the most appropriate use of inhibitors of the biosynthesis of the DNA of the herpes virus. The DNA polymerase of herpes virus is a key enzyme in the replication cycle of the virus and, therefore, is the most attractive target for suppressing the propagation of the virus. Currently, the main group of drugs used to treat HSV, are nucleoside analogs, precursors to inhibitors of the DNA polymerase of herpes virus. These include first of all acyclic nucleoside analogues: ACV, valacyclovir (malinowy ester of acyclovir), and ganciclovir, penciclovir and famciclovir.

Previously patented postit ACV (A. C. 1594953 of the USSR, 1990), as a compound showing significant antiherpetic activity on the strain that is resistant to the action AZW.

However, it has not expressed, which may be due to its anionic nature.

The invention consists in obtaining new nonionic phosphate derivatives ACV as compounds vasoconstrictive suppress reproduction In efticiency compositions.

In accordance with the present invention offers a non-ionic esters of phosphite and alkoxycarbonylmethyl ACV (formula I and II):

where R' R''=alkyl, aryl or aralkyl

These phosphonate esters are not charged compounds that can effectively penetrate the cell membrane and act as a depot form ACV with prolonged action.

The mechanism for the transformation of compounds of formula I and II in the active form inside cells to date is not clear. Main the proposed mechanism is that there is a direct or sequential chemical or enzymatic cleavage phosphonate group with the formation of ACV and its subsequent phosphorylation to the triphosphate. These compounds can be relevant for chemotherapy of herpes infections and can be used as medicines. To obtain pharmaceutical compositions based on compounds of formulas I and II may use various tools (fillers) and organic or inorganic nature, which are used in the production process of finished dosage forms to give them the necessary properties. The content of Cote prepared by known technologies.

To obtain the compounds of formulas I and II can be used in the following synthetic approach:

where R' R''=Alk-, Ar-, Ar-(CH2)n-

He is condensation ACV with phosphor or alkoxycarbonyl-phosphonic acids followed by esterification charged phosphonates III and IV corresponding alcohol. The resulting compounds of formulas I and II can be isolated and purified routine methods, for example, precipitation, chromatography, and so on

The present invention is illustrated by the following examples.

Example 1. Synthesis of P-(ISO-propyl ether) phosphite of acyclovir (I, R=iPr) and its selection.

To a cooled to 0°With the solution phosphite ACV (III) (71 mg, 0.23 mmol) in a mixture of 5 ml of pyridine and 0.5 ml of ISO-propanol was added PivCl (100 μl, 0.8 mmol), kept for 18 h at 5°C. the reaction was Diluted mass of 10 ml of water, was evaporated in vacuum, sopariwala with water (3×5 ml), the residue was dissolved in 2 ml of water and was applied to a column (2×18 cm.) with LiChroprep RP-8 (Merck, 25-40 μm) was suirable in a linear gradient Meon in water (020%, 0.5 V l). The fractions containing phosphonate I, evaporated, pererestorani in water and freeze-dried. Yield 43 mg (56%).1H-NMR (DMSO-d6, , ppm, J, Hz): 10.62 is.T (2H, J 4.4, CH2OS), 1.23 t (6N, JCH3, CH6.2, CH3-iPr).31P-NMR (DMSO-d6): 8.16 FGD (1JH,P698,3JP, CH2~3JR, CH9.2). UV (methanol): max254 nm.

Example 2. Synthesis of P-(ISO-propyl ether) ethoxycarbonylphenyl of acyclovir (II, R'=Et, R''=iPr, and its selection.

To a cooled to 5°C the solution ethoxycarbonylphenyl ACV (IV, R'=Et) (76 mg, 0.2 mmol) in a mixture of 5 ml of pyridine and 0.5 ml isopropanol was added with stirring two portions with an interval of 3 h 2,4,6-triisopropylbenzenesulfonyl (120 mg, 0.4 mmol), and then kept for 18 h at 5°C. the reaction was Diluted mass of 10 ml of water, was evaporated in vacuum, sopariwala with water (3×5 ml), the residue was dissolved in 3 ml of water and was applied to a column (2×18 cm) with LiChroprep RP-8, was suirable in a linear gradient Meon in water (030%, 0.5 V l). The fractions containing phosphonate II, was evaporated, persuasively with ethanol, dissolved in a mixture of chloroform and methanol (95:5), and was applied to a column (2.5×25 cm) with silica gel, the product was suirable system chloroform - methanol (9:1). The solvent was evaporated in vacuo, the residue was dissolved in water and freeze-dried. Yield 43 mg (56%).1H-NMR (CD3OD): 7.87 (1H, H-8), 5.49 (2H, CH2Gua), 4.79 m (1H, CH-iPr), 4.31 m (4H, CH2'OR, CHP, CH2~3JP, CH7.1).

UV (Meon): max254 nm.

Example 3. Tablet form.

For the preparation of tablets using the following composition: compound I (R''=iPr) 200 mg calcium carbonate precipitated 110 mg, microcrystalline cellulose 40 mg, Aerosil - 13 mg, calcium stearate - 12 mg.

Compound I (R''=iPr) is mixed with Aerosil and microcrystalline cellulose, and add calcium carbonate. The resulting mixture optivault the calcium stearate and mix to obtain a homogeneous mass. This tablet the mixture is pressed into briquettes on a tablet press. These briquettes are crushed in a ball mill. The granules are compressed on a tablet press to obtain tablets weighing 375 mg

They studied the stability of the synthesized compounds of formulas I and II in aqueous solutions and in the serum of a person.

Example 4. Chemical hydrolysis of the compounds of formulas I and II.

10 mm solution of compounds I and II in water (5 μl) is added to 95 µl PBS buffer (pH 7.2) (8.0 g NaCl, 0.2 g KN2PO4, 0.2 g KCl and 2.16 g of Na2HPO4×7 H2About 1 l of water) and incubated at 37°C. After a certain period of time selected aliquots and explore them by TLC and In the private buffer (pH 4.8), buffer B: - 66% of the Meon. 0-5 min with Buffer A; 5-10 min 010% buffer B; 10-25 min 1025% buffer B; 25-30 min 2580% buffer B; 30-35 min 80100% buffer B; 35-40 min of 100% buffer B. the Data shown in table 1.

Example 5. The hydrolysis of compounds of formula I and II in the serum of a person.

10 mm solution of compounds I and II in water (5 ál) add the serum of human blood (100%, 95 μl) and incubated at 37°C. After a certain period of time to aliquot (15 μl) was added Meon (45 μl), incubated 20 min at -20°C and centrifuged. The supernatant evaporated. To the residue was added 10 μl of 5 mm sodium phosphate buffer (pH 4.8) and analyze HPLC under the conditions described above. The results are shown in Table 1.

Example 6. The antiviral activity of phosphonates ACV against HSV-1.

The culture of Vero cells (kidney cells of the African green monkey), support in the environment of the Needle containing 5% of fetal serum of calves.

The reference strain of the herpes virus simple VAC-1/L2received from the State collection of viruses Institute of Virology. D. I. Ivanovsky RAMS. Clinical isolates of HSV-1 resistant ACV (Rth), obtained by infection of cell culture Vero vaccinated material from patients with RR and 1:1, with the addition of 2% fetal calf serum. Cultivation of cells and their subsequent infection is carried out in 96-well plastic plates.

Antivirus action is judged by the ability of the studied compounds to inhibit the development virusinduced cytopathogenic actions (JRC), 50% (ID50) and 95% (ID95compared with the JRC in the control infected cultures. The multiplicity of infection of 0.1 PFU/cell. The results take into account in 48 hours

Cytotoxicity of compounds quantitatively determined by staining cells Trifanova blue. The magnitude CD50accept that the concentration at which cell survival after 72 h of contact with the studied compounds is 50%. Index selectivity IS calculated as the ratio of CD50to ID50.

Thus, it is shown that the tested compounds of formula I and II have the ability to inhibit the reproduction of herpes simplex virus type 1 in cell culture Vero, and the effectiveness of these drugs exceeds the efficiency used in clinical practice drug ACV (table 2) and act on the strains of the herpes simplex virus may be resistant to the creation of new medicines, necessary for treatment of herpes infections.

1. Phosphonate derivatives of 9-[(2-hydroxyethoxy)methyl]guanine (ACV) General formula

where R' R'' represent alkyl, aryl or aralkyl.

2. The method of obtaining compounds on p. 1, involving the condensation ACV with phosphor or alkoxycarbonylmethyl acids, esterification charged phosphonates corresponding alcohols, with subsequent isolation and purification of routine methods, for example by precipitation, chromatography and so on

3. Connection on p. 1 with selective Antiherpes virus effect of activity with efficiency exceeding the efficiency of ACV, and acting on the strains of the herpes simplex virus that are resistant to the action AZW.

4. Connection on p. 3, included in the effective therapeutic dose of the active substance in the pharmaceutical composition for use as drugs having Antiherpes virus effect of antiviral activity.



 

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