Patent Application
20100292260
The invention relates to 6-vinyl pyrimidine pyrimidinone derivatives and the use thereof as medicament in particular for the treatment of HIV infections, the use thereof for preparing pharmaceutical compositions and methods for the preparation thereof.
Acquired immunodeficiency syndrome (AIDS) is a disease of viral origin for which so far no appropriate therapy has been developed. More than twenty years have passed since the US Center for Disease Control (CDC) reported AIDS for the first time (Gallo, R. C. Science 2002, 298, 1728.); a few years later HIV was identified as the aetiological agent of this pathology. Within a short space of time, AIDS assumed epidemic proportions throughout the world, to the point that today more than 40 million people are infected with AIDS and more than 3 million deaths from this disease were reported in 2003 alone (WHO (UNAIDS), June 2004).
In 1996, the development of highly active antiretroviral therapy (HAART) and the use of a cocktail of drugs had a significant impact on both the treatment of patients and the development of the disease in various countries. Nevertheless, most people infected by HIV/AIDS do not have access to antiretroviral therapy and to the most common chemotherapies used due to the prohibitive costs (Fauci, A. S. Nat. Med. 2003, 9, 839.).
HIV belongs to the class of retroviruses, i.e. those viruses in which the genetic information is carried by the RNA (Turner, B. G.; Summers, M. F. J. Mol. Biol. 1999, 285, 1.). HIV infects the T cells that carry the antigen CD4 on the surface. In particular, the virus infection requires the fusion of the viral and cellular membranes; this process is mediated by the viral glycoproteins of the capside (gp120, gp41) and by the receptors (CD4 and coreceptors, such as CCR5 or CXCR4) of the target cell. When the virus enters the cell, the RNA thereof is reverse-transcribed to DNA through a viral enzyme, reverse transcriptease (RT). The viral DNA is then integrated into the DNA of the host cell through the entry of HIV. Activation of the host cell is translated into transcriptease of the viral DNA in m-RNA, which is in turn transformed into viral glycoproteins. The protease of HIV, the third viral enzyme, occurs in the phase in which the precursors of the viral glycoproteins are broken down into mature single proteins.
RNA and the viral glycoproteins assemble at the level of the cell surface to form new virions that are released outside the cell to infect other cells. The extensive cell damage arising from the destruction of the host's genetic material and from the release of the virions leads to the death of the infected cells.
There currently exist three classes of antiretroviral drugs approved by the FDA for the treatment of HIV/AIDS. These drugs are reverse transcriptease inhibitors (RTIs), protease inhibitors (PIs) and, recently, entry inhibitors.
The RTIs can be further subdivided into nucleoside inhibitors (NRTIs) and non nucleoside reverse transcriptease inhibitors (NNRTIs). The NRTIs are simply modified nucleosides without the hydroxy group in position 3′ which, once the nucleosides have been incorporated into the DNA, entail the termination of the transcription of the DNA. The anti-HIV activity of these compounds depends on the intracellular phosphorylation thereof and on the capacity of the phosphorylated molecules to interact with the RT of HIV-1. The greater limits to use of the similar nucleosides are due to the toxicity, to the early development of resistance by the virus. The non nucleoside RT inhibitors (NNRTIs) bind at a allosteric site far from the polymerisation site, causing a conformational variation of the active site of the RT that translates into inhibition of the action thereof. The NNRTIs can be further distinguished from the NRTIs by the characteristic resistance to mutations and lack of activity in relegation to HIV-2. On the basis of current knowledge, two strains of HIV are known: HIV-1 and HIV-2. The former is found mainly in Europe, America and Central Africa. HIV-2, on the other hand, is found more commonly in West Africa and Asia and causes a milder clinical syndrome than the former strain. Currently, there are no drugs approved by the FDA for treating HIV-2 and the diagnostic test used for HIV-1 is not applicable to HIV-2 and it is very difficult to isolate it from the blood. In addition, HIV-2 infections develop more slowly. The infections are often asymptomatic and as much as 15-20 years may pass before the infection manifests itself in the blood. The transmission paths are the same as for HIV-1.
The other critical phase of the viral cycle of the virus is the proteolysis of the protein polypeptide precursors and mature enzymes. All the protease inhibitors (PIs) that are currently commercially available such as anti HIV/AIDS are non-hydrolysable peptidomimetics in which the peptide bond is replaced by an isoster (statin, norstatin, hydroxyethylene).
The enfuviride (T-20) is a synthetic peptide and is the first compound that is active as an entry inhibitor to have been introduced into therapy. The enfuviride stops the entry of the virus into the host cell by interfering with the fusion process. This single-action mechanism makes T-20 active against forms of HIV-1 that are resistant to the aforementioned three other classes of antiretroviral drugs (NRTIs, NNRTIs and PIs). On the basis of the clinical data, it can be concluded that long-term suppression of the virus by T-20 is possible if the latter is used in combination with the other antiretroviral drugs.
The non nucleoside RT inhibitors are one of the most important classes of compounds for the treatment of HIV-1 infections. Nevertheless, the development of more active compounds that are more resistant to mutations and are less toxic is required.
The authors of the present invention have synthesised compounds with a general structure 1 and 2 (Botta et al. J. Comb. Chem. 2005, 7, 117):
R1=R2=ethyl, propyl, butyl, propargyl
R1=methyl and R2=benzyl
Ar=phenyl, p-fluorophenyl
m-trifluoromethyl, o-methoxy
that comprises the compound MB3B.
In the present invention, other compounds have been synthesised that show an inhibiting activity of the RT of HIV-1 that is greater than MB3B and a different action mechanism. The interest in new synthesised compounds has developed above all through the fact that one of the different derivatives obtained has an inhibiting activity in relation to the RT of HIV-1 that is 3 orders of magnitude greater than that of MB3B, opening up a new branch of research into the functionalisation of this latest derivative.
The object of the present invention is a compound of general formula I or II
in which:
R1 and R2 represent independently H, ethyl, methyl, propyl, butyl, pentyl, propargyl and allyl;
X represents H, I, Cl, Br, methyl, propyl or substituted alkyl, aryl or aralkyl substituted groups;
Z represents CH2, O, NH.
R3 represents H or an aryl with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl, substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is —H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
R6 represents Y—R7,
or in
which:
Y═S, SO or SO2,
R7 represents methyl, ethyl, propyl, butyl, pentyl, cyclopentyl, cyclohexyl, or aryl substituted groups with the formula:
in which R6′, R7′, R8′, R9′, R10′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, m is comprised between 0 and 4;
R7 also represents substituted cinnamoils with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which V is N, S, O and R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6 alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, m is comprised between 0 and 4;
R4 and R5 represent independently H, methyl, ethyl, benzyl, cyclopentyl, allyl, propargyl, pentyl, aryl substituted groups with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
R4 and R5 represent independently H, methyl, ethyl, benzyl, cyclopentyl, allyl, propargyl, pentyl, aryl substituted groups with the formula
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
W is C1-6 alkyl or amine;
— represents a single or double bond;
W′ is C1-6 alkyl substituted groups, having optionally one or more heteroatoms being, but not only, O, N, or S;
or a pharmaceutically acceptable salt, prodrug or tautomer thereof;
in which the formula I compounds with the following substitutes are not included:
R1═R2=ethyl, propyl, butyl or propargyl, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═S or SO2 and R7=methyl, and
R1=methyl, R2=benzyl, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═S or SO2 and R7=methyl;
The compound preferably has the formula I in which R1═R2═CH3, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═SO2, R7═CH3.
The compound preferably has the formula I in which R1═R2═CH3, X═I, Z═CH2, R3═H, R6═Y—R7 in which Y═SO2, R4═CH3.
The compound preferably has the formula I in which R1═R2═CH2—CH3, X═I, Z═CH2, R3═H, R6═Y—R7 in which Y═SO2, R7═CH3;
The compound preferably has the formula I in which R1═R2═CH2—CH3, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═SO2, R7=p-methoxybenzyl;
The compound preferably has the formula I in which R1, R2, R3 and X═H, Z═CH2, R6═Y—R7 in which Y═SO2, R7═CH3.
The compound preferably has the formula I in which R1═R2═H, X═CH3, Z═O, R3=p-fluorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula I in which R1═R2═H, X═CH3, Z═O, R3=or-dichlorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula I in which R1═R2═H, X═CH3, Z═O, R3=or-difluorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula I in which R1═R2═CH2—CH3, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula I in which R1═R2═CH3, X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula I in which R1═R2═CH3, X═H, Z═CH2, R3═H,
in which R4═H, R5=benzyl.
The compound preferably has the formula I in which R1═R2═CH3, X═H, Z═CH2, R3═H,
in which R4=methyl, R5=ethyl.
The compound preferably has the formula I in which R1═R2═CH3, X═H, Z═CH2, R3═H,
in which R4═H, R5=p-cyanophenyl.
The compound preferably has the formula II in which:
X═H, Z═CH2, R3═H, R6═Y—R7 in which Y═SO2, R7═CH3.
The compound preferably has the formula II in which X═CH3, Z═O, R3=p-fluorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula II in which X═CH3, Z═O, R3=or-dichlorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula II in which X═CH3, Z═O, R3=or-difluorophenyl, R6═Y—R7 in which Y═S, R7=p-methoxybenzyl.
The compound preferably has the formula II in which X═H, Z═CH2, R3═H,
in which R4═H and R5=benzyl.
The compound preferably has the formula II in which X═H, Z═CH2, R3═H,
in which R4=methyl and R5=ethyl.
The compound preferably has the formula II in which X═H, Z═CH2, R3═H,
in which R4═H and R5=p-cyanophenyl.
Still another object of the present invention is a compound of formula III or IV,
in which:
R1 and R2 represent independently H, ethyl, methyl, propyl, butyl, pentyl, propargyl and allyl;
X represents H, I, Cl, Br, methyl, propyl or alkyl groups, aryl or aralkyl substituted groups;
R3 represents H or an aryl with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
R6 represents Y—R7,
in which:
Y═S, SO or SO2,
R7 represents methyl, ethyl, propyl, butyl, pentyl, cyclopentyl, cyclohexyl, or aryl substituted groups of formula:
in which R6′, R7′, R8′, R9′, R10′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, m is comprised between 0 and 4;
R4 and R5 represent independently H, methyl, ethyl, benzyl, cyclopentyl, allyl, propargyl, pentyl, aryl substituted groups with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl groups, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
W is C1-6 alkyl or amine;
— represents a single or double bond;
W′ is C1-6 alkyl substituted groups, having optionally one or more heteroatoms being, but not only, O, N, or S;
or a pharmaceutically acceptable salt, prodrug or tautomer thereof.
Another object of the present invention is a compound of formula V:
in which:
X represents H, I, Cl, Br, methyl, propyl or alkyl groups, aryl or aralkyl substituted groups;
R3 represents H or an aryl with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
R6 represents Y—R7,
in which:
Y═N, S, SO or SO2,
R7 represents substituted cinnamoils with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups;
R7 also represents substituted systems with the formula:
in which V is N, S, O and R11′, R12′, R13′, R14′, R15′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, m is comprised between 0 and 4;
R4 and R5 represent independently H, methyl, ethyl, benzyl, cyclopentyl, allyl, propargyl, pentyl, aryl substituted groups with the formula:
in which R1′, R2′, R3′, R4′, R5′ are independently H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, aryl substituted groups, halo, haloalkyl (in particular CF3), OCH3, NO2, CN, CONH2, CONH—C1-6alkyl, CON(C1-6alkyl)2, NH2, NH—C1-6alkyl, N(C1-6alkyl)2, NHC(O)alkyl, NHSO2—C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, SO2N(C1-6alkyl)2, OZ′ or SZ′ where Z′ is H, or alkyl, aryl or aralkyl substituted groups, n is comprised between 0 and 4;
W is C1-6 alkyl or amine;
— represents a single or double bond;
W′ is C1-6 alkyl substituted groups, having optionally one or more heteroatoms including, but not only, O, N, or S;
R8 represents methyl, ═O, ═CH2;
or a pharmaceutically acceptable salt, prodrug or tautomer thereof.
An object of the present invention is a pharmaceutical composition comprising a pharmaceutically effective and acceptable quantity of a compound of general formula I, II, III, IV or V. The composition preferably comprises at least another compound having an anti-HIV activity.
Still another object of the present invention is, the use of the compound of general formula I, II, III, IV or V for preparing a medicament with antiviral activity. The activity is in particular anti-HIV.
The object of the present invention is a method for the preparation of a compound of general formula I or II comprising the phases shown in diagram 1 or 2 or 3 or 4.
Another object of the present invention is method for the preparation of a compound of general formula III or IV comprising the phases shown in diagram 5.
Another object of the present invention is method for the preparation of a compound of general formula V comprising the phases shown in diagram 6, 7 or 8.
The present invention will now be disclosed by way of non-limiting example with particular reference to the following figures:
The general structure compounds I and II in which R4═CH3, R3═H, Z═CH2 and R6═Y—R7 in which Y═S, SO, SO2, R7═CH3 (6, 7, 8, 9a, 11a) are synthesised with the procedure shown in Diagram 1.
The general structure compound 13 is known in the literature and synthesised according to previously reported methods (Botta, M.; Occhionero, F.; Saladino, R.; Crestini, C.; Nicoletti, R. Tetrahedron Lett. 1997, 38, 8249-8252). The synthesis method for obtaining the compounds 8 with X═H and 11 a is shown in Botta et al. J. Comb. Chem. 2005, 7, 117.
For the compounds in which X═I, Cl, Br a general procedure was used that was previously devised in our laboratories for the halogenation of functionalised pyrimidinones (Paolini, L.; Petricci, E.; Corelli, F.; Botta, M Synthesis 2003, 1039-1044.).
In order to obtain the general structure compounds I in which R6═Y—R7 with Y═SO2 and R7 other than CH3 the procedure shown in Diagram 2 was used.
The procedure for synthesising the product 18 was developed after several attempts starting with the product 16 through sulphonic oxidation 17 and subsequent substitution with NaHS; other commonly used reagents for obtaining products with a correlated structure have not given satisfactory results in the case of this type of derivative. Functionalisation of the product 18 thus continues for treatment with a suitable halogen derivative in a microwave oven in the presence of K2CO3, subsequent dehydration (6′) and oxidation of the S with OXONE® at 0° C. to obtain selectively sulphoxide (Y═SO) or at ambient temperature to isolate the sulphone (Y═SO2).
In order to obtain the derivatives with a general formula II in which Z═O, diagram 3 has been followed.
In order to obtain products with a general structure I and II with
another synthetic strategy was used that replaces the methyl group in position two with a suitable amine through a “one-pot” reaction in which the S atom is first oxidised with m-chloroperbenzoic acid and is then substituted by a suitable amine (Diagram 4).
The proposed procedure is the only one that provided satisfactory results for our purposes. Once the replaced product 21 has been obtained the ester is reduced to alcohol and procedure is conducted in the same conditions for the synthesis of the compounds 15, 16, 6 and 9a, respectively.
The procedure was applied to compounds with Z═CH2, R3═H but those skilled in the art will be able to apply it to compounds with different Z and R3. In fact, in the case of the compounds with Z═CH2, R3═H is the most complex chemically both as a synthesis procedure and as functionalisation in the different phases.
In order to obtain products with a general structure III in which R6═Y—R7 the synthetic procedure shown in diagram 5 was followed:
The suitable cycled product 20, obtained with the classic procedures found in the literature for the synthesis of similar compounds (Box, V. G. S.; Marinovich, N.; Yiannikouros, G. P. Heterocycles 1991, 2, 245-251.), was subjected to complete chloridisation through microwave treatment with SOCl2 in DMF and is then transformed into corresponding carbonyl derivative for H2O treatment at reflux over 12 hours. The obtained derivative was then treated with a suitable amine in order to obtain the functionalisation in C4 and was then subjected to a Wittig reaction, supplying the vinil derivative 21 with good yields. Through cyclopropanation of the double bond the compound 22 was obtained that was then functionalised in C2 to supply the derivatives III for treatment with suitable benzyl halides substituted in DMF, in the presence of K2CO3 in a microwave oven for 5 minutes (Petricci, E.; Mugnaini, C.; Radi, M.; Corelli, F.; Botta, M. J. Org. Chem. 2004, 69, 7880-7887.). The corresponding sulphoxides and sulphides of the derivative 23 were obtained through treatment of the latter with OXONE® at 0° C. and ambient temperature respectively.
For the synthesis of the general formula derivatives III with
The derivative 24 was treated at reflux with suitable amines.
The general structure compounds V are synthesised with the procedure indicated in Diagram 6, 7 or 8.
Method A:
Preparation of the Compound 14a:
1H-NMR (CD3OD): δ (ppm) 3.93-3.87 (t, J=6.67, 2H); 3.06-2.99 (t, J=6.67, 2H); 2.54 (s, 1H).
MS: m/z 313 [M+1]+; 335 [M+Na]+; 646 [2M+Na]+.
Preparation of the Compound 14b:
1H-NMR (CD3OD): δ (ppm) 3.94-3.89 (t, J=6.49, 211); 3.02-2.95 (t, J=6.49, 2H); 2.55 (s, 3H).
m.p.: 168-170° C.
MS: m/z 243 [M+Na]+.
Preparation of the Compound 14c:
1H-NMR (CD3OD): δ (ppm) 3.92-3.87 (t, J=6.50, 2H); 3.03-2.97 (t, J=6.50, 2H); 2.55 (s, 3H).
m.p.:166-169° C.
MS: m/z 266 [M+1]+.
Preparation of the Compounds 15(a-d):
in which for 15a (X═I), 15b (X═Cl) and 15e (X═Br) and 15d (X═H)
15a: X═I (yield 73%)
1H-NMR (CDCl3): δ (ppm) 7.95-7.83 (d, J=8.00, 2H); 7.46-7.38 (d, J=8.00, 2H); 3.92-3.88 (t, J=6.78, 2H); 2.89-2.77 (t, J=6.78, 2H); 2.44 (s, 3H); 2.35 (s, 3H).
m.p.:122-124° C.
MS: m/z 466 [M+1]+; 488 [M+Na]+.
15b: X═Cl (yield 70%)
1H-NMR (CDCl3): δ (ppm) 7.92-7.85 (d, J=8.50, 2H); 7.44-7.37 (d, J=8.50, 2H); 3.95-3.91 (t, J=6.45, 2H); 2.86-2.76 (t, J=6.45, 2H); 2.41 (s, 3H); 2.32 (s, 3H).
m.p.: 94-95° C.
MS: m/z 398 [M+Na]+.
15c: X═Br (yield 31%)
1H-NMR (CDCl3): δ (ppm) 7.95-7.87 (d, J=7.97, 2H); 7.42-7.35 (d, J=7.97, 2H); 3.97-3.90 (t, J=5.70, 2H); 2.86-2.79 (t, J=5.70, 2H); 2.39 (s, 3H); 2.35 (s, 3H).
MS: m/z 458 [M+1]+; 442 [M+Na]+.
15d: X═H (yield 92%)
1H-NMR (CDCl3): δ (ppm) 7.77-7.73 (d, J=8.46, 2H); 7.22-7.18 (d, J=8.46, 2H); 6.46 (s, 1H); 3.81-3.75 (t, J=5.70, 2H); 2.77-2.71 (t, J=5.70, 2H); 2.29 (s, 3H); 2.19 (s, 3H).
MS: m/z 341 [M+1]+; 363 [M+Na]+.
Preparation of the Compound 16:
in which X═I, Cl, Br, H; R1═R2=ethyl, H; R1═H and R2=pentyl, propargyl, allyl.
Analytical data on certain synthesised compounds:
16a: X═I, R1═R2=ethyl (yield 98%)
1H-NMR (CDCl3): δ (ppm) 3.96-3.90 (t, J=5.30, 2H); 3.58-3.47 (q, J=7.14, 4H); 3.00-2.95 (t, J=5.30, 2H); 2.44 (s, 3H); 1.23-1.16 (t, J=7.14, 6H).
MS: m/z 368 [M+1]+; 390 [M+Na]+.
16b: X═I, R1═H, R2=pentyl, (yield 49%)
1H-NMR (CDCl3): δ (ppm) 3.97-3.81 (t, J=5.40, 2H); 3.52-3.42 (m, 2H); 2.92-2.87 (t, J=5.40, 2H); 2.54 (s, 3H); 1.64-1.54(m, 2H); 1.36-1.29(m, 4H); 0.93-0.86 (m, 3H).
m.p.: 73-75° C.
MS: m/z 382 [M+1]+; 404 [M+Na]+.
16c: X═I, R1═H, R2=propargyl, (yield 74%)
1H-NMR (CDCl3): δ (ppm) 4.29-4.26 (m, 2H); 3.97-3.95 (m, 2H); 2.95-2.91 (t, J=5.00, 2H); 2.49 (s, 3H); 2.26-2.23 (m, 1H).
m.p.: 134-136° C.
MS: m/z 350 [M+1]+; 372 [M+Na]+.
16d: X═I, R1═H, R2=allyl, (yield 100%)
1H-NMR (CDCl3): δ (ppm) 6.02-5.81 (m, 1H); 5.27-5.13 (m, 2H); 4.16-4.10 (m, 2H); 4.10-3.96 (m, 2H); 2.96-2.90 (m, 2H); 2.52 (s, 3H).
m.p.: 74-76° C.
MS: m/z 352 [M+1]+; 374 [M+Na]+.
16e: X═Cl, R1═H, R2=pentyl, (yield 67%)
1H-NMR (CDCl3): δ (ppm) 3.95-3.88 (m, 2H); 3.50-3.44 (m, 2H); 2.88-2.81 (t, J=5.40, 2H); 2.51 (s, 3H); 1.75-1.52 (m, 2H); 1.34-1.27 (m, 4H); 0.91-0.85 (m, 3H).
m.p.: 64-67° C.
MS: m/z 290 [M+1]+; 312 [M+Na]+.
16f: X═Cl, R1═H, R2=propargyl, (yield 37%)
1H-NMR (CDCl3): δ (ppm) 4.31-4.28 (m, 2H); 3.99-3.94 (m, 2H); 2.94-2.90 (t, J=5.00, 2H); 2.48 (s, 3H); 2.26-2.23 (m, 1H).
m.p.: 114-116° C.
MS: m/z 258 [M+1]+; 280 [M+Na]+.
16g: X═Cl, R1═H, R2=allyl, (yield 79%)
1H-NMR (CDCl3): δ (ppm) 6.02-5.81 (m, 1H); 5.27-5.13 (m, 2H); 4.16-4.10 (m, 2H); 4.10-3.96 (m, 2H); 2.96-2.90 (m, 2H); 2.52 (s, 3H).
m.p.: 74-76° C.
MS: m/z 280 [M+Na]+.
16h: X═Br, R1═H, R2=pentyl, (yield 57%)
1H-NMR (CDCl3): δ (ppm) 3.95-3.84 (t, J=5.40, 2H); 3.52-3.42 (m, 2H); 2.93-2.89 (t, J=5.40, 2H); 2.54 (s, 3H); 1.64-1.54 (m, 2H); 1.36-1.29 (m, 4H); 0.93-0.86 (m, 3H).
MS: m/z 335 [M+1]+; 357 [M+Na]+.
16i: X═Br, R1═H, R2=propargyl, (yield 65%)
1H-NMR (CDCl3): δ (ppm) 4.33-4.28 (m, 2H); 4.00-3.95 (m, 2H); 2.93-2.90 (t, J=5.00, 2H); 2.48 (s, 3H); 2.28-2.20 (m, 1H).
m.p.: 113-115° C.
MS: m/z 303 [M+1]+; 325 [M+Na]+.
16l: X═Br, R2=allyl, (yield 62%)
1H-NMR (CDCl3): δ (ppm) 5.99-5.85 (m, 1H); 5.27-5.15 (m, 2H); 4.16-4.10 (m, 2H); 4.10-3.96 (m, 2H); 2.94-2.92 (m, 2H); 2.48 (s, 3H).
m.p.: 63-65° C.
MS: m/z 328 [M+Na]+.
Preparation of the Compound 16(m-p):
in which R1═R2=methyl and for 16m: X═I, for 16n: X═H, for 16o: X═Cl and for 16p: X═Br;
Analytical data on certain synthesised compounds:
16m: X═I, R1═R2=methyl (yield 77%)
1H-NMR (CDCl3): δ (ppm) 3.98-3.85 (t, J=5.39, 2H); 3.11 (s, 6H); 3.09-2.95 (t, J=5.39, 2H); 2.48 (s, 3H).
MS: m/z 340 [M+1]+.
16n: X═H, R1═R2=methyl (yield 50%)
1H-NMR (CDCl3): δ (ppm) 5.97 (s, 1H); 3.98-3.84 (t, J=5.40, 2H); 3.09 (s, 6H); 2.81-2.72 (t, J=5.40, 2H); 2.47 (s, 3H).
MS: m/z 214 [M+1]+; 236 [M+Na]+.
Preparation of the Compound 6:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl.
Analytical data on certain synthesised compounds:
6a: X═I, R1═R2=methyl (yield 40%)
1H-NMR (CDCl3): δ (ppm) 7.14-7.00 (dd, Jcis=16.6, Jgem=3.35, 1H); 6.54-6.44 (?, 1H); 5.56-5.50 (?, 1H); 3.09 (s, 6H); 2.51 (s, 3H).
m.p.:59-61° C.
MS: m/z 322 [M+1]+.
6b: X═I, R1═R2=ethyl (yield 40%)
1H-NMR (CDCl3): δ (ppm) 7.15-7.01 (m, 1H); 6.51-6.41 (dd, Jcis=13.02, Jgem=2.54, 1H); 5.53-5.47 (dd, Jcis=12.02, Jgem=2.54, 1H); 3.46-3.38 (m, 4H); 2.48 (s, 3H); 1.71-1.53 (m, 4H); 0.99-0.82 (t, J=7.10, 6H).
MS: m/z 378 [M+1]+.
6c: X═H, R1═R2=methyl (yield 45%)
1H-NMR (CDCl3): δ (ppm) 6.48-6.28 (m, 2H); 5.99 (s, 1H); 5.47-5.42 (d, J=10.4, 1H); 3.05 (s, 6H); 2.49 (s, 3H).
MS: m/z 196 [M+1]+.
Preparation of the Compound 8:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl.
Analytical data on certain synthesised compounds:
8a: X═H, R1═R2=methyl (yield 57%)
1H-NMR (CDCl3): δ (ppm) 6.57-6.46 (m, 2H); 6.34 (s, 1H); 5.61-5.55 (dd, Jcis=9.82, Jgem=1.85, 1H); 3.26 (s, 3H); 3.13 (s, 6H).
m.p.:197° C. dec
MS: m/z 250 [M+Na]+; 477 [2M+Na]+.
8b: X═I, R1═R2=methyl (yield 57%)
1H-NMR (CDCl3): δ (ppm) 7.20-7.07 (dd, Jcis=9.89, Jgem=6.76, 1H); 6.64-6.55 (d, J=6.7, 1H); 5.71-5.65 (d, J=9.89, 1H); 3.29 (s, 3H); 3.24 (s, 6H).
m.p.:197° C. dec.
MS: m/z 354 [M+1]+; 376 [M+Na]+; 729 [2M+Na]+.
Preparation of the Compound 17:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl.
Analytical data on certain synthesised compounds:
17: X═H, R1═R2=ethyl (yield 50%)
1H-NMR (CDCl3): δ (ppm) 6.35 (s, 1H); 4.74 (bs, 1H); 4.01-3.95 (t, J=5.78, 2H); 3.52-3.44 (m, 4H); 3.23 (s, 3H); 2.91-2.85 (t, J=5.78, 2H); 1.22-1.15 (t, J=6.8, 6H).
MS: m/z 274 [M+1]+; 296 [M+Na]+; 312 [M+K]+.
Preparation of the Compound 18:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl.
Analytical data on certain synthesised compounds:
18: X═H, R1═R2=ethyl (yield 55%)
1H-NMR (CD3OD): δ (ppm) 6.12 (s, 1H); 3.82-3.70 (m, 3H); 3.55-3.44 (m, 4H); 2.68-2.62 (t, J=6.06, 2H); 1.21-1.14 (t, J=7.79, 6H).
MS: m/z 228 [M+1]+; 250 [M+Na]+; 476 [2M+Na]+.
Preparation of the Compound 19:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl, Y=pentyl, p-methoxybenzyl.
Analytical data on certain synthesised compounds:
19a: X═H, R1═R2=ethyl, Y=p-methoxybenzyl chloride (yield 95%)
1H-NMR (CDCl3): δ (ppm) 7.45-7.28 (m, 2H); 6.89-6.79 (m, 2H); 5.89 (s, 1H); 5.25 (s, 2H); 3.90-3.84 (t, J=5.6, 2H); 3.48-3.41 (m, 4H); 3.73 (s, 3H); 2.76-2.69 (t, J=5.6, 2H); 1.21-1.11 (t, J=8.00, 6H).
MS: m/z 348 [M+1]+; 370 [M+Na]+.
19b: X═H, R1═R2=ethyl, Y=pentyl (yield 95%)
1H-NMR (CDCl3): δ (ppm) 5.81 (s, 1H); 3.81-3.75 (t, J=5.10, 2H); 3.41-3.31 (q, J=6.88, 4H); 2.63-2.57 (t, J=5.10, 2H); 2.05-2.02 (m, 5H); 1.59-1.50 (m, 4H); 1.08-1.017 (t, J=6.88, 6H).
MS: m/z 296 [M+1]+; 318 [M+Na]+.
Preparation of the Compound 6′:
in which X═I, Cl, Br, H; R1═R2=methyl, ethyl, H; R1═H and R2=pentyl, propargyl, allyl, R4=pentyl, p-methoxybenzyl.
6′a : X═H, R1═R2=ethyl, R4=p-methoxybenzyl (yield 52%).
1H-NMR (CDCl3): δ (ppm) 7.34-7.29 (d, J=8.10, 2H); 6.89-6.85 (d, J=8.10, 2H); 6.82-6.78 (m, 2H); 5.98 (s, 1H); 5.53-5.47 (d, J=10.4, 1H); 4.60 (s, 1H); 3.87-3.76 (m, 5H); 1.19-1.11 (t, J=8.00, 6H).
MS: m/z 330 [M+1]+; 352 [M+Na]+.
6′b: X═H, R1═R2=methyl, R4=p-methoxybenzyl (yield 40%).
1H-NMR (CDCl3): δ (ppm) 7.35-7.31 (d, J=8.58, 2H); 6.81-6.77 (d, J=8.58, 2H); 6.59-6.45 (dd, J=17.31, J=9.83, 1H); 6.41-6.31 (dd, J=17.31, J=9.83, 1H); 6.01 (s, 1H); 5.50-5.44 (dd, J=9.83, J=2.15, 1H); 4.35 (s, 2H); 3.75 (s, 3H); 3.06 (s, 6H).
MS: m/z 302 [M+1]+; 324 [M+Na]+.
Preparation of the Compound 9a:
9a: yield 56%
1H-NMR (CDCl3): δ (ppm) 6.51-6.45 (m, 1H); 6.07 (s, 1H); 5.62-5.55 (m, 2H); 2.64 (s, 3H).
MS: m/z 191 [M+Na]+.
Preparation of the Compound 21:
21 a: R4═H; R5=benzyl; yield 68%
1H-NMR (CDCl3): δ (ppm) 7.32-7.26 (m, 5H); 5.44 (s, 1H); 4.55-4.53 (d, J=2.76, 2H); 4.23-4.12 (q, J=7.08, 2H); 3.37 (s, 2H); 1.25-1.19 (t, J=7.08, 3H).
MS: m/z 288 [M+1]+; 310 [M+Na]+.
Preparation of the Compound 26a
The 3-(4-methoxyphenyl)-propenol (0.3 mmol) is suspended in anhydrous DMF (1 mL) in the presence of trimethylphosphine (0.45 mL) (1 M solution in toluene) and left for 10 minutes. The reaction mixture is taken to 0° C. and CBr4 (0.15 g, 0.45 mmol) is added. The mixture is irradiated in a microwave oven at 40° C. for 5 min and the appropriate thiouracil (0.3 mmol) is then added, synthesised as previously reported by Botta, Corelli et al. (J. Med. Chem. 2005, 48, 8000-8008). The mixture is irradiated at 130° C. for 5 min and is then diluted with water (2 mL) and extracted with diethyl ether (5×10 mL). Finally, the combined organic phases were dried on anhydrous Na2SO4 and evaporated. The combined organic phases are dried on anhydrous Na2SO4 and evaporated. The residue is purified by chromatographic flash to provide a solid that is then recrystallised.
Yield 75%. Mp 211-212° C. IR (CHCl3) (ν, cm−1): 1539, 1660, 3002. 1H NMR (DMSO-d6): δ 2.03 (s, 3H), 3.47 (d, 2H, J=7.20 Hz), 3.72 (s, 3H), 4.18 (s, 2H), 5.62-5.73 (m, 1H, J=7.20 Hz, Jtrans=15.49 Hz), 6.24 (d, 1H, Jtrans=15.49 Hz), 6.85-6.89 (m, 2H), 7.13-7.44 (m, 5H). MS (ESI) m/z: 447 [M+H]+, 469 [M+Na]+. HPLC (C8 column; CH3OH/H2O, 80/20) tR 6.34 min.
Preparation of the Compound 27a:
The p-nitroiodobenzene (2.14 mmol) and the propargyl alcohol are suspended in the minimum quantity of DMF. The following are added in order to this solution: triethylamine (4.28 mmol), PdCl2(PPh3)2 (0.21) and CuI (0.65 mmol) the solution is subjected to magnetic stirring at ambient temperature for 5 minutes. It is diluted with water (2 mL) and it is extracted with ethyl acetate (3×10 mL). The combined organic phases are dried on anhydrous Na2SO4 and evaporated. The residue is purified by chromatographic flash to provide a solid that is then recrystallised.
Yield 79%. Mp 95-96° C. IR (CHCl3) (ν, cm−1): 1522, 3031, 3609. 1H NMR (CDCl3): δ 4.52 (s, 2H), 7.55 (d, 2H, Jortho=8.72 Hz), 8.16 (d, 2H, Jortho=8.72 Hz). MS (ESI) m/z: 178 [M+H]+.
The 3-(4-Nitrophenyl)-propinol (0.3 mmol) is suspended in anhydrous DMF (1 mL) in the presence of trimethylphosphine (0.45 mL) (1 M solution in toluene) and left for 10 minutes. The reaction mixture is taken to 0° C. and CBr4 (0.15 g, 0.45 mmol) is added. The mixture is irradiated in a microwave oven at 40° C. for 5 min and then the appropriate thiouracil (0.3 mmol) is added, synthesised as previously reported by Botta, Corelli et al. (J. Med. Chem. 2005, 48, 8000-8008). The mixture is irradiated at 130° C. for 5 min and is then diluted with water (2 mL) and extracted with diethyl ether (5×10 mL). Finally, the combined organic phases are dried on anhydrous Na2SO4 and evaporated. The combined organic phases are dried on anhydrous Na2SO4 and evaporated. The residue is purified by chromatographic flash to provide a solid that is then recrystallised.
Yield 67%. IR (CHCl3) (ν, cm−1): 1643, 2931, 3368. 1H NMR (DMSO-d6): δ 2.02 (s, 3H), 3.93 (s, 4H), 6.95-6.99 (m, 1H), 7.15-7.35 (m, 2H), 7.51 (d, 2H, Jortho=8.51 Hz), 8.18 (d, 2H, Jortho=8.51 Hz). MS (ESI) m/z: 428 [M +H]+. HPLC (C8 column; CH3OH/H2O, 80/20) tR 4.10 min.
Preparation of the Compound 28a:
A solution of 2-bromoethanol (12.8 mmol) and K2CO3 (12.8 mmol) in anhydrous DMF (2 mL) is subjected to magnetic stirring at ambient temperature for 10 minutes. The 4-methoxybenzyl chloride (12.8 mmol) is added and is left at 60° C. for 2h. It is diluted with water (2 mL) and is extracted with ethyl acetate (3×10 mL). The combined organic phases are dried on Na2SO4 anhydrous and evaporated. The residue is purified by chromatographic flash to provide a colourless oil.
Yield 54%. 1H NMR (CDCl3): δ 3.45 (t, 2H, J=5.83 Hz), 3.73 (t, 2H, J=5.83 Hz), 3.85 (s, 3H), 4.55 (s, 2H), 6.85 (d, 2H, Jortho=8.16 Hz), 7.25 (d, 2H, Jortho=8.16 Hz).
The appropriate thiouracil (0.3 mmol) and 1′1(2-bromoettossimetil)-4-metossibenzene (0.3 mmol) are suspended in anhydrous DMF in the presence of K2CO3 (0.3 mmol), and the mixture is left at ambient temperature for 2 h. It is diluted with water (2 mL) and is extracted with ethyl acetate (3×10 mL). The combined organic phases are dried on Na2SO4 anhydrous and evaporated. The residue is turified by chromatographic flash to provide a colourless oil.
Yield 70%. Mp 138-140° C. IR (CHCl3) (ν, cm−1): 1644, 2958. 1H NMR (DMSO-d6): δ 1.99 (s, 3H), 2.85 (t, 2H, J=5.82 Hz), 3.12 (t, 2H, J=5.82 Hz), 3.71 (s, 3H), 4.10 (s, 2H), 4.20 (s, 2H), 6.83-6.87 (m, 2H), 7.12-7.36 (m, 5H), 12.51 (br s, 1H). MS (ESI) m/z: 465 [M+H]+, 487 [M +Na]+. HPLC (C8 column; CH3OH/H2O, 80/20) tR 6.24 min.
Biological Activity
Enzyme Sample
In a final volume of 25 microlitres the following reagents were mixed: 50 mM Tris-HCl pH 7.5, 0.25 mg/ml BSA, 0.5 mM DTT, 20-50 nM HIV-1 (recombinant) reverse transcriptase, nucleotide substrate dTTP marked radioactively (3H, 4 Ci/mmol) and nucleic acid (poly(rA)/oligo(dT)). The concentrations of dTTP and poly(rA)/oligo(dT) in standard conditions were 5 μM and 0.5 μM, respectively. In the experiments shown in
The reaction mixture is incubated 10 min. at 37° C. and 20 μl are removed and deposited on a GF/C Whatman glass fibre filter measuring 25 mm in diameter. The filters are washed 3 times for 5 min. in 5% trichloroacetic acid and once for 5 min. in absolute alcohol. After being dried, the filters are immersed in a scintillating mixture (Packard) and the acid precipitable radioactivity is measured by a scintillation counter for β-emitting isotopes (Beckman).
In some experiments, the HIV-1 recombinant reverse transcriptase was substituted by a reverse transcriptase containing the mutations K.103N and Y181I. They were produced by cloning the gene of the mutated RT in an expression vector for prokariotic cells according to the method reported in the literature (Maga G, Amacker M, Ruel N, Hubscher U, Spadari S. Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions. J Mol Biol. Dec. 19, 1997; 274(5):738-47).
The dose-response curves obtained by means of samples of enzyme activity in the presence of increasing doses of inhibitor were analysed according to the equation E(%)=Emax/(1-(I/ID50), where E(%) is the fraction of enzyme activity that is measurable in the presence of each concentration of inhibitor, Emax is the activity in the absence of an inhibitor, I is the concentration of inhibitor. The ID50 was calculated by analysing data using the GraphPad Prism graphic interpolation programme (for Macintosh).
Test of Inhibition of Viral Proliferation on Culture Cells
The biological activity of the compounds was evaluated on lymphoid cell lines MT-4 against the wild strain of HIV-1 NL4-3. The MT-4 cells were briefly infected with the appropriate HIV-1 or the infection was simulated to determine the cytoxicity, in the presence of different concentrations of potential inhibitor compounds. 5 days after the infection a colorimetric method was used that involves using a tetrazole salt (MTT) to evaluate the number of surviving cells. The mutant IRLL98 HIV-1 contains the following mutations in the coding sequence for RT:M41L, D67N, Y181C, M184V, R211K, T215Y (resistance to NRTI) and the mutations K101Q, Y181C, G190A (resistance to NNRTI). The H1V-1 mutants containing multi-NNRTI mutations, K103N or Y188L as mutants were supplied by the Medical Research Council Centralised Facility for AIDS Reagents, Herfordshire, UK.
Biological Results
Compound 8a
The compound 8a proved to be the most active of the chemical series that we synthesised and has shown a reverse transcriptase ID50 that is 3 orders of magnitude greater than that of the previously synthesised compound MB3B and a particular action mechanism compared with the compounds known hitherto in the literature.
Studies of the action mechanism of the compound 8a have shown that the molecular structure thereof is the binary complex RT:DNA and that inhibition of reverse transcriptase is competitive with the nucleotide. These characteristics are different from those of the classic non-nucleoside inhibitors, the target of which is the enzyme that is free of the substrates and the bond of which does not influence the subsequent link of the nucleic acid and nucleotide substrates.
The demonstration of the action mechanism of 8a is based on three distinct experimental observations:
1. The inhibition of the RT induced by 8a decreases with the increase of the concentrations of TTP in the reaction. By performing titration of 8a in a reverse transcription reaction in vitro with recombinant RT and in the presence of different concentrations of nucleotide substrate (in this case dTTP) a decrease in the capacity thereof to inhibit RT is shown, as is observed from the increase in the M50 values shown in
2. The apparent affinity (Kapp) of the RT for dTTP decreases as the concentration of 8a increases. Equally if titrations of nucleotide substrate (dTTP) are taken in a reverse transcription reaction in vitro with recombinant RT and in the presence of different concentrations of 8a, there is a reduction in the affinity of the RT for the substrate, as shown by the increase of apparent constant affinity (Kapp), in
3. The inhibition of the RT induced by 8a increases the concentration of nucleic acid poly(rA)/oligo(dT) (generally known as DNA although it is an RNA-DNA hybrid). If titrations 8a are made in a reverse transcription reaction in vitro with recombinant RT and in the presence of different concentrations of substrate (in this case poly(rA)/oligo(dT)) an increase of the capacity thereof to inhibit RT is observed, as shown by the progression of the curves shown in
In view of the fact that RT binds the substrates in the order: 1) nucleic acid, 2) dNTP; with the increase in the concentration of DNA, the free enzyme becomes saturated with DNA and only afterwards does the binary complex RT:DNA bind to dNTP to form the ternary complex. The datum in
It is thus possible to conclude that 8a preferentially binds to the binary complex of the RT with the nucleic acid and that this bond prevents the subsequent interaction with the nucleotide substrate.
Other Derivatives
On the basis of the results obtained for 8a other compounds have been synthesised that show an activity as inhibitors of the RT of HIV-1 and an action mechanism that is similar to 8a. Table 1 shows the results of the enzyme tests conducted on some of the synthesised compounds.
As shown in Table 1, seven different positions (R1-R7, Z, X and Y) of the molecule of formula I have been considered in which R6═Y—R7. The optimal position was found with the derivative 8a, which shows an increase in activity in relation to the wild type enzyme of 2900 times. It is further also significantly active against enzymes containing significant resistance mutations in a clinical environment such as those in positions 103 and 181. On the other hand, the derivative 8a did not show appreciable activity in tests of inhibition of viral proliferation in culture cells (Table 1). The activity of the compound 25a at cell level is significantly worthy of note: not so much the inhibition of the wt as the maintenance of activity on mutated strains of the enzyme such as K103N and Y181L is of interest. It is these latter data that make the study of this new class of pyrimidine derivatives of further interest.
As shown in Table 2, the compounds 26a-r generally have an excellent inhibiting activity against the wild-type strain of reverse transcriptase (WT), both in enzyme and cell tests.
In particular, the compounds 26a, 26e, 26g, 26l, 26n, 26p, 26q showed nanomolar and subnanomolar activity. As far as the activity of the compounds 26a-r on mutant strains is concerned, enzyme tests have enabled lead compounds to be identified in the compounds 26a, 26n, 26p, which lead compounds are interesting for designing further active compounds on the mutant K103N. These compounds in fact showed micromolar and submicromolar activity in cell tests on both the mutant K103N and on the other mutants examined (Y181C, Y188L).
The compounds 27a-c shown in Table 3, which respectively show the similar ones of the compounds 26i, 26g and 26a performing an alkyne rather than an alkene function on the lateral chain bound to the C2 of the pyrimidinone core, showed a decrease in inhibitory activity in both cell and enzyme tests.
This decrease in activity can thus be correlated to the loss of important interactions with the aromatic portion bound to the lateral chain in C2 following the introduction of a planar linear system like the alkyl system.
| Number | Date | Country | Kind |
|---|---|---|---|
| RM2005A000512 | Oct 2005 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IT2006/000734 | 10/13/2006 | WO | 00 | 6/25/2010 |
