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WO1996010565A1 - Substituted 6-benzyl-4-oxopyrimidines, process for their preparation and pharmaceutical compositions containing them - Google Patents

Substituted 6-benzyl-4-oxopyrimidines, process for their preparation and pharmaceutical compositions containing them Download PDF

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Publication number
WO1996010565A1
WO1996010565A1 PCT/EP1995/003912 EP9503912W WO9610565A1 WO 1996010565 A1 WO1996010565 A1 WO 1996010565A1 EP 9503912 W EP9503912 W EP 9503912W WO 9610565 A1 WO9610565 A1 WO 9610565A1
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compounds
reacted
preparation
alkyl
hiv
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PCT/EP1995/003912
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French (fr)
Inventor
Marino Artico
Silvio Massa
Antonello Mai
Paolo La Colla
Maria Elena Marongiu
Enzo Tramontano
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Istituto Superiore Di Sanita'
Universita' Degli Studi Di Cagliari
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Priority to AU38036/95A priority Critical patent/AU3803695A/en
Publication of WO1996010565A1 publication Critical patent/WO1996010565A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/56—One oxygen atom and one sulfur atom

Definitions

  • the present invention refers to compounds having general formula (I):
  • X is selected from the group consisting of 0 and S;
  • R is selected from the group consisting of C 1-4 alkyl and C 5-6 cycloalkyl
  • AIDS acquired immunodeficiency syndrome
  • the transcription phase of the viral genome (a single RNA filament) in double strand DNA is the most studied one.
  • RT reverse transcriptase
  • DABO 3,4-dihydro-2-alkoxy-6- benzyl-4-oxopyrimidines
  • X is selected from the group consisting of 0 and S;
  • R is selected from the group consisting of C 1 -4 alkyl and C 5-6 cycloalkyl
  • the compounds of the present invention differ from the DABO described in the above reported literature owing to the presence of one S atom in the place of the 0 atom or owing to the presence of substituents on the benzylic ring.
  • Tables 3 and 4 the activity of some compounds according to the invention is reported, while in the Table 5 the data obtained with the above mentioned DABO compounds are reported by comparison.
  • methyl iodide (8 mmol; 1.13 g) is added to a solution containing the suitable 2-thiouracil derivative (4 mmol) dissolved in anhydrous N,N-dimethylformamide (2 ml) and the mixture is left under agitation at room temperature until the starting material disappears by the thin-layer chromatography check (silica gel/n-hexane: ethyl acetate: methanol 12:3:1).
  • the solution is diluted with water (200 ml), the aqueous phase is extracted with ethyl acetate (3 x 50 ml) and the reunited organic extracts are washed with a solution saturated with sodium thiosulfate (100 ml), with a solution saturated with NaCl (100 ml), dried (Na 2 SO 4 ) and deprived of the solvent.
  • anhydrous potassium carbonate (4.2 mmol) and the suitable alkyl halide (4.4 mmol) are added to a solution containing the suitable 2-thiouracil derivative (4 ml) dissolved in anhydrous N,N-dimethylformamide (2 ml) and the resulting mixture is left under agitation at room temperature (method B) or at 80 °C (method C) until the starting material disappears by the thin-layer chromatography check (silica gel/n-hexane: ethyl acetate: methanol 12:3:1).
  • the solution is diluted with water (200 ml), it is acidified to pH 5 with 0.5 N acetic acid and the aqueous phase is extracted with ethyl acetate (3 x 50 ml).
  • the reunited organic extracts are washed with a saturated solution of sodium thiosulfate (100ml), with a saturated solution of NaCl (100 ml), dried (Na 2 SO 4 ) and concentrated at reduced pressure.
  • the agitation has been continued for 1 hour at 0 °C and for a further hour at room temperature, then the mixture has been poured on ice and treated with 2N HCl.
  • the organic layer has been picked up and the aqueous solution washed two times with CH 2 CI 2 .
  • the organic phase and the extracts have been reunited, washed with brine and dried.
  • the mixture has been diluted with water (100 ml) after cooling, acidified to pH 5 with 0.5 N acetic acid and extracted with ethyl acetate (3 x 50 ml).
  • rRT recombinant enzyme
  • the cells used in this study were MT-4 and C8l66, both T4 lymphocytes lines permissive for the HIV replication.
  • the cells were suspended in RPMI 1640 added with fetal calf serum (FCS) at 10%, penicillin 100 U/ml and streptomycin 100 ⁇ g/ml.
  • FCS fetal calf serum
  • the cell cultures were incubated at 37 °C in 5% CO 2 atmosphere and were periodically checked to verify the absence of mycoplasmas contamination.
  • cytotoxicity a colorimetric method has been employed based on the use of a tetrazolium salt, the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide (MTT), which is transformed by the mitocondrial enzyme succinic dehydrogenase into a blue coloured product, the formazane, the amount of which turns out to be directly proportional to the number of viable cells.
  • MTT 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide
  • the amount of formazane was then determined at the spectrophotometer by evaluation of the optical density at 570 nm.
  • the values shown in the columns CC 50 represent the compound concentrations required to reduce by 50% the MTT metabolization and, therefore, the cell viability; the mitocondrial metabolic process is, in fact, in a linear relation with the cell viability.
  • the major part of the compounds has low or null cytotoxicity in non infected cells, even at the maximum concentrations tested.
  • the inhibition of the virus-induced cytopathogenicity constituted the estimation criterion of the anti-HIV- 1 activity of the compounds.
  • the virus used in the antiviral tests (HIV-1, strain III B ) has been obtained from the chronically infected H9/III B cells supernatant.
  • the virus stock solutions were titled in C8166 and mantained at -80 °C till the moment of use.
  • MT-4 cells seeded at a density equal to 1 x 10 /ml, were infected with HIV-1 at a multiplicity of infection (m.o.i.) equal to 0.01. After 1 hour of incubation at 20 °C and subsequent removal of the inoculum, the cells were washed three times and then suspended again at a density equal to 1 x 10 ⁇ /ml, in absence or in presence of the test compounds.
  • the cell survival was determined with the above mentioned MTT method, in order to compute the values of EC 50 representing the compound concentration necessary to reduce by 50% the virus-induced cytopathogenicity.
  • test compounds are active in inhibiting the HIV-1 multiplication in MT-4 cells. They, owing to the lack of citotoxicity, have a selectivity index (meant as ratio between cytotoxicity and anti-HIV activity) particularly favourable.
  • RT reverse transcriptase
  • the gene of this enzyme has been formerly cloned in an expression vector, the protein has been expressed in E.coli and subsequently has been purified to obtain a preparation with a high purity degree.
  • the tests with the recombinant RT (rRT) have been carried out at 37 °C for 30 minutes in 50 ⁇ l containing 50 mM tris-HCl (pH 7.8), 1 mM dithiotreitol, 80 mM KCl, 6 mM MgCl 2 .
  • a unit is defined as the amount of enzyme necessary to incorporate 1 nmol of [ 3 H]-dTTP in the "template-primer" Poly(rA)-oligo(dT) 10 in one minute at 37 °C. After incubation, 40 ⁇ l of the reaction have been transferred on Whatman GF/A glass fiber filters and processed for the determination of the acid insoluble radioactivity after treatment with trichloroacetic acid. The values reported in the Tables (IC 50 ) represent the compound concentration required to reduce the enzyme activity by 50%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)

Abstract

Substituted 6-benzyl-4-oxopyrimidines having formula (I) are described, wherein: X is selected from the group consisting of O and S; R is selected from the group consisting of C1-4 alkyl and C5-6 cycloalkyl; R', R' and Z, equal or different among them mean H or C1-4 alkyl considering that, when X = O, R and R' cannot be both equal to H; their pharmaceutically acceptable salts and their soluble derivatives; one of their preparation processes and their use for the preparation of pharmaceutical compositions useful for the treatment of viral infections.

Description

SUBSTITUTED 6-BENZYL-4-OXOPYRIMIDINES, PROCESS FOR THEIR PREPARATION
AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
FIELD OF THE INVENTION
The present invention refers to compounds having general formula (I):
Figure imgf000003_0001
wherein:
X is selected from the group consisting of 0 and S;
R is selected from the group consisting of C1-4 alkyl and C5-6 cycloalkyl;
R', R" and Z, equal or different among them mean H or C1-4 alkyl considering that, when X=O, R and R' can not be both equal to H; their pharmaceutically acceptable salts and their soluble derivatives;
one of their preparation processes and their use for the preparation of pharmaceutical compositions useful for the treatment of viral infections, particularly of immunodeficiency virus (HIV) infections. PRIOR ART
The pandemic diffusion of the acquired immunodeficiency syndrome (AIDS) makes urgent the development of chemotherapeutic agents able to halt the replication of the two retroviruses responsible for the infection: HIV-1 and HIV-2.
Among the various phases characterizing the replication cycle of these viruses the transcription phase of the viral genome (a single RNA filament) in double strand DNA is the most studied one.
Such a phase, taking place early after the infection, is catalyzed by virus specific enzyme, the reverse transcriptase (RT). The products of pharmaceutical interest able to inhibit the RT may be essentially divided into two classes: nucleosides analogues and non-nucleoside compounds. The four drugs used until now in the AIDS therapy, i.e. AZT, ddl, dhT and ddC, belong to the first class. Other molecules having very different chemical structure, some of which are undergoing clinical trials, belong to the second one. The 3,4-dihydro-2-alkoxy-6- benzyl-4-oxopyrimidines (DABO) structurally similar to the compounds according to the present invention and having antiviral properties are described in Antiviral Chemistry and Chemotherapy (1993) 4(6), pp. 361- 368. Unfortunately the clinical experience has pointed out two major limits of the therapy with said antivirals.
Following chronical treatment, on the one hand collateral toxicity phenomena appear (remarkable in the case of the nucleosides analogous). On the other hand, drug-resistant mutants appear (very quickly in the case of non-nucleoside RT inhibitors). It is therefore evident the necessity to have available always new molecules active and useful in this field of application.
DETAILED DESCRIPTION OF THE INVENTION
The present invention allows to overcome the above mentioned drawbacks by compounds having general formula (I)
Figure imgf000005_0001
wherein:
X is selected from the group consisting of 0 and S;
R is selected from the group consisting of C1 -4 alkyl and C5-6 cycloalkyl;
R', R" and Z, equal or different among them mean or C1-4 alkyl considering that, when X=O, R and R' can not be both equal to H;
their pharmacologically acceptable salts and their soluble derivatives. As it can be noticed, the compounds of the present invention differ from the DABO described in the above reported literature owing to the presence of one S atom in the place of the 0 atom or owing to the presence of substituents on the benzylic ring. In Tables 3 and 4 the activity of some compounds according to the invention is reported, while in the Table 5 the data obtained with the above mentioned DABO compounds are reported by comparison. In the light of the biological activity data, the products having formula (I) wherein:
Figure imgf000005_0002
= 3
3 ,
Figure imgf000006_0001
turned out to be particularly interesting.
PREPARATION OF THE COMPOUNDS HAVING FORMULA (I) WHEREIN X = S (see scheme "A")
Thiourea (43 mmol) and the suitable methyl phenylacetylacetate (31.5 mmol) are added to a solution of sodium methoxide obtained dissolving metallic sodium (0.063 g-atoms) in anhydrous methanol (50 ml) and the resulting mixture is left to reflux under magnetic agitation for 5 hours. After cooling the solvent is evaporated at reduced pressure, the residue is taken back with water (200 ml) and the mixture is acidified to pH 5 with 0.5 N acetic acid and extracted with ethyl acetate (3 x 100 ml).
The solid in case separated (raw 2-thiouracil) is vacuum filtered, stove dried and crystallized by a suitable solvent while the reunited organic extracts are washed with a saturated solution of NaCl (2 x 100 ml), dried (Na2SO4) and concentrated at reduced pressure to give the 2-thio(5-alkyl)-6-benzyl(substituted)uracil (1).
Subsequently, according to the method A, methyl iodide (8 mmol; 1.13 g) is added to a solution containing the suitable 2-thiouracil derivative (4 mmol) dissolved in anhydrous N,N-dimethylformamide (2 ml) and the mixture is left under agitation at room temperature until the starting material disappears by the thin-layer chromatography check (silica gel/n-hexane: ethyl acetate: methanol 12:3:1). Subsequently the solution is diluted with water (200 ml), the aqueous phase is extracted with ethyl acetate (3 x 50 ml) and the reunited organic extracts are washed with a solution saturated with sodium thiosulfate (100 ml), with a solution saturated with NaCl (100 ml), dried (Na2SO4) and deprived of the solvent.
The 3,4-dihydro-2-methylthio-(5-alkyl)-6-benzyl (substituted)-4- oxopyrimidine derivatives (2) so obtained are then purified by a suitable solvent.
Alternatively, according to the methods B and C, anhydrous potassium carbonate (4.2 mmol) and the suitable alkyl halide (4.4 mmol) are added to a solution containing the suitable 2-thiouracil derivative (4 ml) dissolved in anhydrous N,N-dimethylformamide (2 ml) and the resulting mixture is left under agitation at room temperature (method B) or at 80 °C (method C) until the starting material disappears by the thin-layer chromatography check (silica gel/n-hexane: ethyl acetate: methanol 12:3:1).
Subsequently the solution is diluted with water (200 ml), it is acidified to pH 5 with 0.5 N acetic acid and the aqueous phase is extracted with ethyl acetate (3 x 50 ml). The reunited organic extracts are washed with a saturated solution of sodium thiosulfate (100ml), with a saturated solution of NaCl (100 ml), dried (Na2SO4) and concentrated at reduced pressure.
The 3,4-dihydro-2-alkylthio-(5-alkyl)-6-benzyl (substituted) -4- oxopyrimidine derivatives (3) and (4) so obtained are then purified by crystallization from a suitable solvent or by chromatography (silica gel/n-hexane: ethyl acetate: methanol 12:3:1). The physico-chemical data of some of the obtained products are reported in the Table 1.
PREPARATION OF THE COMPOUNDS HAVING GENERAL FORMULA (I) WHEREIN X = 0 (see scheme B)
SOCI2 (21.3 ml) is slowly added under nitrogen atmosphere to the suitable phenylacetic acid (43.2 mmol) and the resulting solution has been warmed for 2 hours. After cooling the solvent has been dried at reduced pressure.
A solution of the raw 3' -methyl or 3' »5' -dimethyl phenylacetyl chloride (160 mmol) so obtained in 50 ml of anhydrous CH2CI2 has been added in 2 hours, at 0 °C and under nitrogen atmosphere, to a suspension of 23-75 g (165 mmol) of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum acid) in 65 ml of anhydrous CH2CI2 containing 32.5 ml (400 mmol) of anhydrous pyridine, under strong agitation. The agitation has been continued for 1 hour at 0 °C and for a further hour at room temperature, then the mixture has been poured on ice and treated with 2N HCl. The organic layer has been picked up and the aqueous solution washed two times with CH2CI2. The organic phase and the extracts have been reunited, washed with brine and dried.
The evaporation of the solvent under reduced pressure gave the acylated product 5 as a brown solid which has been put to reflux in 200 ml of CH3OH for 20 hours.
After vacuum evaporation of the solvent and chromatographic purification the compounds 6 are respectively obtained.
Metallic sodium (3*68 g) is added to a solution of the above mentioned compounds 6 in methanol (250 ml) and the solution is stirred to complete dissolution of the metal. CHoI is dropped in the solution and the resulting mixture is reflux warmed for 4 hours.
After cooling the solvent has been removed and the residue has been treated with H2O (200 ml) and extracted with CCl 3 (3 x 100 ml). The organic layer has been washed with brine (2 x 100 ml), dried and evaporated to give a residue which, purified by chromatography, has given the compounds 7 as a yellow oil.
A solution of the compounds 6 or 7 (10 mmol) in CH3OH (50 ml) has been added to a suspension of 0-methylisourea hydrogensulfate (15 mol) and Ca(OH)2 (16 mmol) under strong agitation. The resulting mixture has been stirred at room temperature for 12 hours and then concentrated, acidified to pH 5 with 0.5 N acetic acid and extracted with ethyl acetate (3 x 50 ml). The organic extracts have been washed with brine (100 ml), dried and dry evaporated. The residue, purified by crystallization from a suitable solvent gave the pure compounds 8.
Metallic potassium (100 mmol) in small pieces has been slowly added under agitation to the suitable alcohol (200 ml) freshly distilled on sodium. The dissolution of the metal has been completed by warming the mixture at 70-80 °C and then the derivatives 8 (10 mmol) are added and the obtained mixture is reflux warmed under nitrogen atmosphere. The reaction has been stopped when the chromatographic check confirmed the disappearance of the starting 4-pyrimidone.
The mixture has been diluted with water (100 ml) after cooling, acidified to pH 5 with 0.5 N acetic acid and extracted with ethyl acetate (3 x 50 ml).
The reunited extracts have been washed with brine (100 ml), dried and evaporated to give the raw products 9 which have been purified by column chromatography and crystallized again by a suitable solvent.
In some cases the methoxy group in the position 2 of the compounds 8 wherein R = R2 = H, R1 = CH3 or R = R1 = R2 = CH3 has been removed with formation of the respective compounds 10 wherein R = R1 = H, R2 = CH3 and R = R1 = R2 = CH3 as collateral products.
The physico-chemical data of some of the obtained products are reported in the Table 2.
The products obtained acting as above described with the relative data of cytotoxicity and anti-HIV 1 activity are reported in the Tables 3 and 4.
BIOLOGICAL ACTIVITY
In order to illustrate the activity of the compounds in the HIV-1 infections the results in vitro are reported relating to:
- cytotoxicity for different cell lines and bone marrow cells from HIV seronegative subjects;
- inhibitory activity with regard to HIV-1;
- capability to inhibit the reverse transcriptase of HIV-1 in tests with recombinant enzyme (rRT) of HIV-1. The cells used in this study were MT-4 and C8l66, both T4 lymphocytes lines permissive for the HIV replication. The cells were suspended in RPMI 1640 added with fetal calf serum (FCS) at 10%, penicillin 100 U/ml and streptomycin 100 μg/ml.
The cell cultures were incubated at 37 °C in 5% CO2 atmosphere and were periodically checked to verify the absence of mycoplasmas contamination.
For the evaluation of the compounds cytotoxicity a colorimetric method has been employed based on the use of a tetrazolium salt, the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide (MTT), which is transformed by the mitocondrial enzyme succinic dehydrogenase into a blue coloured product, the formazane, the amount of which turns out to be directly proportional to the number of viable cells.
In short 50 μl of RPMI containing 1 x 104 cells (MT-4, C8166, U937. PBL) were added, in 96 wells multiplates, to 50 μl of RPMI containing or not scalar dilutions of the compounds under examination. After 4 days of incubation at 37 °C 20 μl of MTT (2.5 μg/ml) have been added to each well. After 4 hours of incubation at 37 °C the produced formazane was solubilized by adding 150 μl/well of an isopropanol solution containing 0.34% of HCl and 5% of Nonidet P40 (NP-40), a non-ionic detergent.
The amount of formazane was then determined at the spectrophotometer by evaluation of the optical density at 570 nm. The values shown in the columns CC50 represent the compound concentrations required to reduce by 50% the MTT metabolization and, therefore, the cell viability; the mitocondrial metabolic process is, in fact, in a linear relation with the cell viability. As it is shown in the Tables, the major part of the compounds has low or null cytotoxicity in non infected cells, even at the maximum concentrations tested. The inhibition of the virus-induced cytopathogenicity constituted the estimation criterion of the anti-HIV- 1 activity of the compounds.
The virus used in the antiviral tests (HIV-1, strain IIIB) has been obtained from the chronically infected H9/IIIB cells supernatant. The virus stock solutions were titled in C8166 and mantained at -80 °C till the moment of use. MT-4 cells, seeded at a density equal to 1 x 10 /ml, were infected with HIV-1 at a multiplicity of infection (m.o.i.) equal to 0.01. After 1 hour of incubation at 20 °C and subsequent removal of the inoculum, the cells were washed three times and then suspended again at a density equal to 1 x 10^/ml, in absence or in presence of the test compounds.
After 4 days of incubation at 37 °C the cell survival was determined with the above mentioned MTT method, in order to compute the values of EC50 representing the compound concentration necessary to reduce by 50% the virus-induced cytopathogenicity.
The results reported in the Tables show that the test compounds are active in inhibiting the HIV-1 multiplication in MT-4 cells. They, owing to the lack of citotoxicity, have a selectivity index (meant as ratio between cytotoxicity and anti-HIV activity) particularly favourable.
In order to complete the antiviral activity analysis of the compounds we proceeded to estimate the effects of the interaction of the various molecules with the target enzyme, the reverse transcriptase (RT). The gene of this enzyme has been formerly cloned in an expression vector, the protein has been expressed in E.coli and subsequently has been purified to obtain a preparation with a high purity degree. The tests with the recombinant RT (rRT) have been carried out at 37 °C for 30 minutes in 50 μl containing 50 mM tris-HCl (pH 7.8), 1 mM dithiotreitol, 80 mM KCl, 6 mM MgCl2. 0.1 mg/ml bovine serum albumin, 10 μM [3H]-dTTP (ICi/mmol) or [3H]-dGTP (1 Ci/mmol), 0.05 OD260 units/ml of Poly(rC)-oligo(dG)12-18 and 0.002 units of enzyme. A unit is defined as the amount of enzyme necessary to incorporate 1 nmol of [3H]-dTTP in the "template-primer" Poly(rA)-oligo(dT)10 in one minute at 37 °C. After incubation, 40 μl of the reaction have been transferred on Whatman GF/A glass fiber filters and processed for the determination of the acid insoluble radioactivity after treatment with trichloroacetic acid. The values reported in the Tables (IC50) represent the compound concentration required to reduce the enzyme activity by 50%.
The analysis of the values of IC50 reveals a good correlation with the values of EC50 confirming that the specific target of the compounds object of the invention is the reverse transcriptase.
Figure imgf000014_0001
Figure imgf000015_0001
X
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001

Claims

1. Compounds having general formula (I)
Figure imgf000021_0001
wherein:
X is selected from the group consisting of 0 and S;
R is selected from the group consisting of C1-4 alkyl and C5-6 cycloalkyl;
R', R" and Z, equal or different among them, mean H or C1-4 alkyl considering that, when X=0, R and R' can not be both equal to H;
their pharmaceutically acceptable salts and their soluble derivatives.
2. Compounds having general formula (I) as claimed in claim 1 wherein X=S.
3. Compounds having general formula (I) as claimed in claim 1 wherein:
Figure imgf000021_0002
Figure imgf000022_0001
4 . Process for the preparation of the compounds having formula (I) as claimed in claim 1 wherein X = S, wherein: the suitable methyl phenylacetylacetate is reacted with thiourea in presence of sodium methoxide and the so oDtained 2-thio(5-alkyl)-6-benzyl (substituted)uracils are reacted with methyl iodide, or with an alkyl halide in a basic medium.
5. Process for the preparation of the compounds having formula (I) as claimed in claim 1 wherein X = 0, wherein: a 3'-methyl or 3',5'- dimethylphenylacetyl chloride is reacted with 2,2-dimethyl-1,3-dioxane- 4,6-dione, the so obtained compound is reacted with CH3I, the so obtained compound (or its precursor) is reacted with O-methyl isourea hydrogensulfate and the obtained product is reacted with the suitable potassium alcoholate.
6. Use of the products as claimed in claim 1 for the preparation of pharmaceutical compositions having antiviral activity.
7. Use as claimed in claim 6 wherein the antiviral activity is an anti- HIV activity.
8. Use as claimed in claim 7 wherein the anti-HIV activity is an anti- HIV-1 activity.
9. A therapeutic method for treating viral infections consisting of the administering to a patient in need thereof a therapeutically effective amount of at least one compound having formula (I) according to claim 1.
PCT/EP1995/003912 1994-10-04 1995-10-04 Substituted 6-benzyl-4-oxopyrimidines, process for their preparation and pharmaceutical compositions containing them WO1996010565A1 (en)

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Cited By (3)

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WO2001007027A3 (en) * 1999-07-22 2001-08-09 Vertex Pharma Pyrimidine derivatives for the treatment of viral diseases
CN100439343C (en) * 2006-08-04 2008-12-03 复旦大学 2-Alkylthio-5-alkyl-6-(1-cyanoarylmethyl) substituted uracil compounds and their preparation methods and uses
CN100519540C (en) * 2007-01-09 2009-07-29 云南大学 S-DABO compound, synthesizing method and usage

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EP0123402A2 (en) * 1983-03-25 1984-10-31 Fujisawa Pharmaceutical Co., Ltd. Pyrimidine derivatives, preparation thereof and use thereof
WO1991018887A1 (en) * 1990-06-06 1991-12-12 Smithkline Beecham Intercredit B.V. Diaminopyrimidine compounds

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