WO2006106169A1 - Phosphorothioates derived from nucleoside analogues for antiretroviral therapy - Google Patents

Abstract

The invention relates to the use of cyclic nucleoside analogue phosphorothioates or acyclic nucleoside derivatives having formula (I) or (IB), which are pharmacologically-active inhibitors of the reverse transcriptase enzyme (RT) and to the use thereof in therapy, particularly for the treatment of infections caused by human immunodeficiency virus (HIV). Said compounds are particularly effective when the RT has combinations of mutations that render HIV resistant to RT-inhibitor drugs.

Description

TITLE

PHOSPHOROTIOATS DERIVED FROM NUCLEOSIDE ANALOGS FOR

ANTIRETROVIRAL THERAPY

FIELD OF THE INVENTION

The present invention relates to phosphorothioate compounds derived from nucleoside analogues (both cyclic and acyclic), which have pharmacological activity as inhibitors of the enzyme retrotranscriptase (RT) and their use in therapy, in particular they are directed to the treatment of infections caused by Human immunodeficiency virus (HIV).

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV) is a spherical retrovirus, about 80-100 nm in diameter, which has a lipid envelope in which surface glycoproteins are located. The HIV genome consists of two strands of single-stranded positive polar RNA, 9.8 kilobases [Coffin et al. (ed.). Retroviruses CoId Spring Harbor Lab. Press, Plainview, USA, 1997]. In 1983, it was identified as the virus that causes acquired immunodeficiency syndrome (AIDS), a disease that today causes the death of more than 3 million people every year.

HIV primarily infects cells of the immune system and for its replication and spread requires a number of cellular factors contributed by the host, in addition to viral proteins that intervene at different stages of the life cycle. HIV proteins constitute therapeutic targets of great interest in the development of effective drugs against viral infection. One of the most important targets is retrotranscriptase (RT), a viral enzyme against which most of the drugs currently available in the clinic act. Retrotranscriptase is the enzyme responsible for the replication of the genomic RNA of the virus [Telesnitsky and Goff in Retroviruses. Coffin et al. (ed.), p. 121-160, CoId Spring Harbor Lab. Press, Plainview, USA, 1997]. It is a multifunctional enzyme with RNA and DNA dependent polymeric DNA activity and endonuclease activity (RNase H). RT inhibitors whose clinical use against HIV infection has been approved can be classified into three large groups according to their chemical structure: nucleoside-like inhibitors, acyclic nucleoside-derived phosphonates and non-nucleoside-like inhibitors (for reviews recent, see Menéndez-Arias. Trenas Pharmacol Sci 2002; 23: 381-388; De Clercq. Nature Rev Microbiol 2004; 2: 704-720). To date, the clinical use of seven nucleoside-like inhibitors has been approved: 3 '-azido-3'-deoxythymidine (AZT, zidovudine), 2', 3'-dideshydro-2 ', 3'-dideoxythymidine (d4T, stavudine ), 2 ', 3'-dideoxyinosine (ddl, didanosine), 2', 3'-dideoxy-3'-thiacitidine (3TC, lamivudine), 2 ', 3'-dideoxycytidine (ddC, zalcitabine), (lS, 4R ) -4- [2-amino-6- (cyclopropylamino) -9H-purin-9-yl] -2- cyclopentene-1-methanol (abacavir), and 2 ', 3'-dideoxy-5-fluoro-3' -tiacitidine (FTC, emtricitabine), in addition to an acyclic phosphonate nucleoside: 9- [2- (phosphonomethoxy) propyl] adenine (tenofovir), and others are in preclinical stages of development (De Clercq. Nature Rev Microbiol 2004; 2: 704-720; Sharma et al. Curr TopMed Chem 2004; 4: 895-919; Otto. Curr Opin Pharmacol 2004; 4: 431-436).

All these compounds must be transformed into their corresponding triphosphorylated derivatives inside the cell. Triphosphorylated RT inhibitors compete with the natural substrates of the DNA synthesis reaction (the dNTPs), but lack the 3'-OH group present in the dNTPs, so once incorporated into the DNA chain being synthesized , the polymerization reaction is blocked.

Some of the above-mentioned inhibitors, such as AZT, have the disadvantage of having a certain degree of toxicity in addition to presenting other adverse side effects, so other antiretroviral agents such as 3'-azido-2 ', 3' have also been used. -dideoxyuridine and its mono, di and triphosphate derivatives (US 4,916,122) and organic thiophosphonates of the formula CH ₃ NH (CH ₂ ) ₃ NH (CH ₂ ) ₃ SPO ₃ H ₂ (US 5,824,664) which partly overcome these drawbacks.

en los que la característica principal es la presencia de un resto azúcar como grupo terminal R y la posibilidad de sustituir algunos oxígenos de la cadena de difosfato por átomos de azufre (grupos W). Desde la implantación de terapias antirretrovirales potentes, basadas en la combinación de inhibidores de la RT y de la proteasa, ha disminuido sensiblemente la mortalidad y morbilidad de la infección por el VIH. Sin embargo, la aparición de virus resistentes al tratamiento antirretroviral constituye un problema importante que compromete el tratamiento de los pacientes a largo plazo. La resistencia a fármacos inhibidores de la RT se adquiere mediante mutaciones en la región del genoma viral que codifica para dicha enzima. En el caso de los análogos a nucleósido y de los fosfonatos derivados de nucleósidos acíclicos, las mutaciones que confieren resistencia a dichos fármacos pueden actuar mediante dos mecanismos diferentes: a. Interfiriendo con la capacidad de la RT del VIH para incorporar los derivados trifosforilados del inhibidor, de modo que éste se incorporaría con menor eficacia catalítica que los correspondientes sustratos naturales (Deval et al. Curr Drug Metabol 2004, 5, 305-316). b. Aumentando la actividad de rescate de iniciadores bloqueados en su extremo 3' por inhibidores análogos a nucleósido o por fosfonatos derivados de nucleósidos acíclicos.On the other hand, the therapeutic effectiveness of active nucleosides depends on the ease with which they pass through the cells and undergo phosphorylation. In this regard, compounds of formula are claimed in US Patent 5,159,067:

in which the main feature is the presence of a sugar moiety as a terminal group R and the possibility of replacing some oxygens in the diphosphate chain with sulfur atoms (groups W). Since the introduction of potent antiretroviral therapies, based on the combination of RT and protease inhibitors, the mortality and morbidity of HIV infection have significantly decreased. However, the appearance of viruses resistant to antiretroviral treatment is an important problem that compromises the long-term treatment of patients. Resistance to RT inhibitory drugs is acquired by mutations in the region of the viral genome that codes for said enzyme. In the case of nucleoside analogs and phosphonates derived from acyclic nucleosides, mutations that confer resistance to such drugs can act by two different mechanisms: a. Interfering with the ability of HIV RT to incorporate the triphosphorylated derivatives of the inhibitor, so that it would be incorporated with less catalytic efficacy than the corresponding natural substrates (Deval et al. Curr Drug Metabol 2004, 5, 305-316). b. Increasing the rescue activity of initiators blocked at its 3 'end by nucleoside-like inhibitors or by acyclic nucleoside phosphonates.

Among the mutations that act through the first mechanism we can mention, in addition to the M184V change, the combination A62V / V75I / F77L / F116Y / Q151M, which is an example of multi-resistance (Shirasaka et al. Proc Nati Acad Sci USA 1995; 92: 2398-2402). Compared to wild (non-mutated) RT, the RTs carrying the changes associated with Ql 5 IM have a lower capacity to incorporate triphosphorylated nucleoside analogues, such as AZT-triphosphate, d4T-triphosphate, ddC-triphosphate and ddA-triphosphate (Ueno et al. J Biol Chem 1995; 270: 23605-23611). On the other hand, the accumulation of resistance mutations to thymidine analogues (TAMs), such as M41L, D67N, K70R, L210W, T215Y / F and K219Q / E, has a clinical impact on resistance to AZT, d4T, ddl , abacavir and tenofovir, consistent with the high ATP-dependent phosphorolytic activity on initiators terminated with nucleoside-like RT inhibitors, presented by RTs carrying such mutations (Meyer et al. Mol CeIl 1999; 4: 35-43; Meyer et al. Antimicrob Agents Chemother 2000; 44: 3465-3472; Boyer et al. J Virol 2001; 75: 4832-4842; Naeger et al. Antimicrob Agents Chemother 2002; 46: 2179-2184).

Carrier HIV isolates have been identified in their RT of the T69S amino acid change, associated with an insertion of two amino acids (typically Ser-Ser, Ser-Gly or Ser-Ala) between positions 69 and 70 of the RT, and at one or several TAMs. These viruses have high levels of resistance to AZT and moderate to other RT inhibitors such as d4T, ddC, ddl and tenofovir, in phenotypic assays carried out with recombinant virus (Winters et al. J Clin Invest 199 8; 102: 1769- 1775; Larder et al. Antimicrob Agents Chemother 1999; 43: 1961-1967; Mas et al. EMBO J 2000; 19: 5752-5761; Lennerstrand et al. Antimicrob Agents Chemother 2001; 45: 2144-2146). These data are consistent with the levels of ATP-dependent phosphorolytic activity presented by the corresponding RTs in the presence of initiators blocked with AZT, d4T, ddA or tenofovir (Mas et al. EMBO J 2000; 19: 5752-5761; Mas et al. J MolBiol 2002; 323: 181-197; Boyer et al. J Virol 2002; 76: 9143-9151; Meyer et al. J Virol 2003; 77: 3871-3877; White et al. Antimicrob Agents Chemother 2004; 48: 992 - 1003). For the detection of this activity it has been shown that in addition to the T69S insertion and mutation, the presence of T215Y was required (Matamoros et al. J Biol Chem 2004; 279: 24569-24577). To date, no inhibitors of ATP-dependent phosphorolytic activity are available, which could be used in clinical practice, and that could be used against strains carrying complexes of resistance mutations to thymidine analogues, or multiresistant strains carrying inserts between codons 69 and 70. Therefore, it is necessary to have compounds that allow to block those resistance mechanisms presented by the RTs carrying mutations that confer a high phosphorolytic activity dependent on ATP. BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that triphosphorylated derivatives of nucleoside-derived RT-inhibitors, in which oxygen has been substituted for sulfur in α phosphate, are substrates of the DNA polymerization reaction catalyzed by HIV RT , and block the subsequent elongation of the DNA chain.

As a main feature, and contrary to what happens with non-modified triphosphorylated derivatives on α phosphate, these compounds cannot be eliminated by those resistant RTs that carry mutations that confer a high phosphorolytic activity dependent on ATP, thus inactivating one of the mechanisms of resistance to RT inhibitors of greater incidence in the clinic.

Thus, one aspect of the present invention is the use of a compound of formula (I) or (IB)

(I) (IB) where n is selected from 0, 1 and 2;

A is a C ₁ -C ₃ substituted or unsubstituted alkyl group,

R is any of the purine or pyrimidine nitrogenous bases; Y

R 'and R "are independently selected from H and a C ₁ -C ₁₀ alkyl group,

R '"is hydrogen, an azide group, or does not exist when there is a double bond between carbons T and 3', the dotted line between carbons 2 'and 3' indicates the possibility of a double bond, or a pharmaceutically salt acceptable, a prodrug or solvate thereof, for the preparation of a medicament for the treatment of a patient infected by the

HIV and / or affected by Acquired ImmunoDefi Science Syndrome (AIDS). In a preferred variant R 'is hydrogen. In another preferred variant R '' is hydrogen. In another preferred variant R '"is hydrogen or an azide group. Compounds where both R' and R" are H. are also preferred.

In another variant the compound is acyclic of formula (I) and group A is the group -CH ₂ CH ₂ - or the group -CH (CH ₃ ) CH ₂ -, that is to say compounds similar to adefovir or tenofovir.

In another variant the compound of formula (IB) is 3'-azido-3'-deoxythymidine-5'- O- (l-thiophosphate) (AZTTPaS).

Preferably, the compounds are used for the treatment of patients exhibiting resistance to human immunodeficiency virus (RT) inhibitor drugs due to RT mutations. Thus, patients may present resistance to any of the drugs zidovudine, stavudine, didanosine, lamivudine, zalcitabine, abacavir, emtricitabine, tenofovir, adefovir or mixtures thereof.

In another aspect the invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or

(IB) as defined above, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof, and at least one pharmaceutically acceptable carrier, adjuvant, or vehicle, for administration to a patient.

A third aspect of the invention is constituted by a compound of formula (I)

(I) where n is selected from 0, 1 and 2; A is a C ₁ -C ₃ substituted or unsubstituted alkyl group, R is any of the purine or pyrimidine nitrogenous bases; Y R 'and R "are independently selected from H and a C ₁ -C ₁ O alkyl group, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof.

BRIEF DESCRIPTION OF THE CONTENT OF THE FIGURES Figure 1. Model representing the mechanism of cleavage of primers terminated with nucleoside-like inhibitors (eg AZT).

Figure 2. (A) Mold-initiator complex used in the study, and position where AZT-triphosphate will be incorporated.

(B) Rescue kinetics of AZT-terminated initiator, in reactions catalyzed by wild RT (BHlO strain) and a multi-drug resistant RT antiretroviral RT

(RT SS). Lanes P and B correspond to the initiator before and after incorporating the inhibitor. The remaining lanes (from left to right) correspond to aliquots collected at 2, 4, 6, 8, 10, 12, 15, 20 and 30 min after the addition of 3.2 mM ATP. The phosphorolytic rescue activity correlates with the amount of fully elongated product.

(C) Rescue kinetics of AZT-terminated primers, obtained in the presence of: (o) ATP; (•) ATPγS.

Figure 3. (A) Incorporation efficiency observed with the S _p diastereoisomer of AZTTPaS, compared to the R _p diastereoisomer of the same compound. P indicates the position of the non-elongated initiator and lanes 1 to 7 correspond to aliquots of the reaction collected after 10 s, 20 s, 30 s, 1 min, 5 min, 15 min and 30 min incubation. (B) Comparison of the rescue efficiency of the RT SS, in the presence of 3.2 mM ATP, on initiators terminated with AZT-monophosphate, or with the products resulting from the incorporation of the S _p and R _p diastereoisomers of AZTTPaS. Lanes P and B correspond to the initiator before and after incorporating the inhibitor. The remaining lanes (from left to right) correspond to aliquots collected at 2, 4, 6, 8, 10, 15 and 30 min after the addition of ATP.

DETAILED DESCRIPTION OF THE INVENTION The compounds employed in the present invention are phosphorylated derivatives of the enzyme retrotranscriptase (RT), responsible for the replication of the genomic RNA of the HIV virus causing AIDS. They have a formula (I) or (IB) as defined above and in the claims. The compounds can be prepared by methods known in the state of the art, such as those described in WO93 / 23415.

The term "pharmaceutically acceptable salts, solvates, prodrugs" refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound that, when administered to a receptor, is capable of providing (directly or indirectly) a compound as described in This document. However, it will be appreciated that pharmaceutically acceptable salts are also within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by methods known in the art.

For example, pharmaceutically acceptable salts of compounds provided herein are synthesized by conventional chemical methods from an original compound containing a basic or acidic moiety. Generally, such salts are prepared, for example, by reacting the free acid or base forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iohydrate, sulfate, nitrate, phosphate and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and salts of organic bases such as, for example, ethylenediamine, ethanolamine, N ₃ N - dialkylene ethanolamine, triethanolamine, glucamine and basic amino acid salts.

Particularly preferred derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by making a compound administered orally more easily absorbed by the blood), or which potentiates the release of the original compound in a biological compartment (for example, the brain or lymphatic system) in relation to the original species. Examples of modifications that can be made to the compounds used in the invention are those described for example in US5,159,067, such as the incorporation of sugar groups on the terminal phosphate, or modifications known as those present in known prodrugs of the drugs zidovudine, stavudine, didanosine, lamivudine, zalcitabine, abacavir, emtricitabine, tenofovir, adefovir and are currently in preclinical or clinical development.

Any compound that is a prodrug of a compound of formula (I) is within the scope of the invention. The term "prodrug" is used in its broadest sense and encompasses those derivatives that become live in the compounds of the invention. Such derivatives will be apparent to those skilled in the art, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the compounds present: esters, amino acid esters, phosphate esters, salts sulfonate esters metallic, carbamates, and amides. Examples of methods for producing a prodrug of a given active compound are known to those skilled in the art and can be found for example in Krogsgaard-Larsen et al. "Textbook of Drug design and Discovery" Taylor & Francis (April 2002).

The compounds employed in the invention may be in crystalline form as free compounds or as solvates and it is intended that both forms are within the scope of the present invention. Solvation methods are generally known within the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.

The compounds employed in the present invention represented by the formula (I) described above may include enantiomers, depending on the presence of chiral centers on a C or on a P, or isomers, depending on the presence of multiple bonds (for example, Z , E). The individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention.

Purine nitrogenous bases refer to natural or synthetic adenine or guanine bases. The nitrogenous pyrimidine bases refer to the natural or synthetic cytosine, thymine or uracil bases. In a preferred embodiment of the invention R 'is hydrogen. In another preferred embodiment R "is also hydrogen when n> 0.

The RT enzyme inhibitors of formula (IB) belong to the so-called nucleoside-like inhibitors and, within their structure, two fundamental aspects must be highlighted: the replacement of an oxygen with a sulfur in the α phosphate; and - the absence of -OH groups in the 3 'position of its ribose ring. RT inhibitors developed to date also lack the 3'-OH group. They are characterized in that they compete with the natural substrates of the DNA synthesis reaction (the dNTPs), but lacking the 3'-OH group, once incorporated into the DNA chain, block the polymerization reaction.

However, it has been observed that resistance to RT inhibitors that exhibit this characteristic can be achieved through the accumulation of mutations in the pol gene of the HIV virus. These mutations can give rise to RTs that have a lower capacity to incorporate the phosphorylated inhibitor into the DNA chain that is being synthesized, or they can act by giving RT an ATP-dependent phosphorolytic activity that allows it to eliminate the inhibitor from end 3 '.

By using the compounds of formula (IB) as defined above, it has been seen that the presence of a sulfur instead of an oxygen as a substitute for the phosphate α of a nucleoside triphosphate, in which the ribose ring lacks of the 3'-OH group, allows the incorporation of the nucleotide in the DNA chain synthesized by RT, but avoids its elimination by ATP-dependent phospholysis, thus inactivating one of the resistance mechanisms to RT inhibitors of greater incidence in the clinic.

Therefore, in a preferred variant of the invention, compounds analogous to any of the compounds zidovudine, stavudine, didanosine, lamivudine, zalcitabine, abacavir, emtricitabine, tenofovir, adefovir where an oxygen has been replaced by a sulfur as a phosphate substituent α are used . Among the RTs present in viruses resistant to antiretroviral drugs, different levels of phosphorolytic activity dependent on ATP have been observed. Table 1 shows those combinations of mutations that confer significant levels. of phosphorolytic activity dependent on ATP. The highest levels of activity have been observed with RTs derived from clinical isolates and carriers of the T69SSS insertion (that is, of the Thr-69 change -> Ser together with the insertion of two serines between positions 69 and 70 of the RT) , accompanied by the change Thr-215 - ^ Tyr and other associated changes in the clinical resistance to AZT (M41L, D67N, L210W, etc).

Table 1. Mutations and combinations of mutations that give rise to RTs with ATP-dependent phosphorolytic activity, and for which their ability to cleave the 3 'end of the initiator to the inhibitors indicated in each case has been demonstrated in vitro.

^a os re er os a es os pa rones e mu ac ones se obtuvieron con RTs derivadas de aislados clinicos, por lo que contienen además otras mutaciones, que en principio no están relacionadas con resistencia a inhibidores de la RT análogos a nucleósid o

^{In these cases, many} were obtained with RTs derived from clinical isolates, so they also contain other mutations, which in principle are not related to resistance to nucleoside-like RT inhibitors or

The rescue of primers terminated with a nucleoside analogue inhibitor involves excision of the onhinitor that blocks the 3 'outside of the DNA being synthesized and its subsequent elongation (Figure 1). RTs that have ATP-dependent phosphorolytic activity are capable of eliminating the various inhibitors used in clinical practice with variable efficacy. Thus, several studies have shown that elimination is more effective on initiators terminated with AZT-monophosphate, followed by those terminated by d4T-monophosphate, and to a lesser extent, ddA-monophosphate (physiological intermediate of didanosine) and tenofovir. A good model system for these studies is the RT called SS, whose detailed description we carried out previously (Mas et al. EMBO J 2000; 19: 5752-5761; Mas et al. J Mol Bio \ 2002; 323: 181 -197). This enzyme is characterized by presenting an amino acid change at position 69 (T69S), accompanied by an insertion of two serines, and also contains a series of additional mutations, including changes M41L, A62V, K70R, Vl 181, M184I, L210W and T215Y. This RT has a very high ATP-dependent phosphorolytic activity and is capable of very effectively cleaving AZT-terminated initiators.

The use of the compounds according to the invention is particularly effective in the case of RTs carrying the following combinations of mutations:

M41L / A62V / T69S / K70R / V118I / M184I / L210W / T215Y, M41L / A62V / D67N / -

T69SSS / K70R / V118I / M184I / L210W / T215Y,

M41L / A62V / T69SSS / K70R / V118I / M184I / L210W / T215Y,

M41L / D67N / K70R / T215 Y / K219Q, M41L / T69S / L74V / L210W / T215 Y, M41L / T69SAG / L210W / R211K / L214F / T215Y,

M41L / T69SSG / L210W / R211K / L214F / T215Y, M41L / T69SSS / L74V / L210W / T215Y, M41L / T69SSS / L210W / R211K / L214F / T215Y, M41L / T69SAG / T215Y15, M41 / T215S15 T215Y, M41L / T215Y, D67N / K70R, D67N / K70R / T215F / K219Q, D67N / K70R / T215Y, D67N / K70R / T215Y / K219Q, T69SSG / T215Y, T69SSS / T215Y, T215FY2, T215F2, T215F2

The rescue reaction involves the formation of a polyphosphate dinucleoside which, in the event that the ribonucleotide used is ATP and the rescued initiator is blocked with AZT-monophosphate, would form a tetraphosphate dinucleoside of AppppAZT structure. It is known that hydrolysis of the phosphodiester bonds between the α and β and β and γ phosphates of ATP (or equivalent ribonucleotide triphosphate) is not necessary for the formation of the corresponding dinucleoside polyphosphate (Meyer et al. Proc Nati AcadSci USA 1998; 95 : 13471-13476). However, the use of adenosine-5'-γ-thiotriphosphate (ATPγS) instead of ATP in the rescue reaction of initiators blocked with AZT-monophosphate allowed us to verify that the rescue rate was reduced to approximately 50% (Figure 2 C; Example 1), demonstrating the need to have in the reaction of all the substituent oxygens of the γ phosphate of the ATP.

On the other hand, it is known that DNA polymerases are capable of incorporating 5'-O- (l-thiotriphosphate) nucleosides derived from adenine, thymine, guanosine and cytosine, although the stereochemistry of the reaction plays an important role in the process. Thus, only the incorporation of the S _p diastereoisomer of the nucleoside 5'-O- (l-thiotriphosphate) is effective, the incorporation of the R _p diastereoisomer being much less effective

(Eckstein. Annu Rev Biochem 1985; 54: 367-402). In addition, the incorporation of the nucleotide involves the inversion of the configuration, so that the Sp diastereoisomer of nucleoside 5'-O- (l-thiotriphosphate) is converted into diastereoisomer R _v in the terminal phosphorothioate of the synthesized DNA (Bartlett and Eckstein. J Biol Chem 1982;

257: 8879-8884).

The incorporation of 3'-azido-3'-deoxythymidine-5'-C> - (l-thiotriphosphate) (AZTTPaS) by HIV RT results in a DNA chain blocked at its 3 'end, thus inhibiting the elongation process The AZTTPaS whose formula responds to the structure:

puede adquirirse a la empresa TriLink Biotechnologies (San Diego, California, EE.UU.), cuyas preparaciones contienen el diastereoisómero S_p en una relación 2 a 1, con respecto al R_v. Ambos diastereoi someros pueden separarse mediante cromatografía en fase reversa (HPLC) utilizando una columna Vydac C₁₈ 218TP54 (4.6 x 250 mm), y eluyéndose en condiciones isocráticas (5 % acetonitrilo en tampón acetato de trietilamonio 10 mM, pH 6,8). En estas condiciones, eluye primero el diastereoisómero Sp y posteriormente el R_v. El catión trietilamonio puede intercambiarse con cationes de sodio haciendo pasar la solución por una resina de intercambio catiónico Dowex 50WX8-200 (Sigma). El ensayo de incorporación y rescate se puede llevar a cabo con un complejo molde-iniciador, cuyo molde lo formaría un oligonucleótido de ADN (por ejemplo, D38, 5 ^'-GGGTCCTTTCTTACCTGCAAGAATGTATAGCCCTACCA-3 ^') y el iniciador sería un oligonucleótido más corto, complementario al anterior (por ejemplo, 25PG5A, 5 ^'-TGGTAGGGCTATACATTCTTGCAGG-3 ^') (Figura 2A). El oligonucleótido de menor tamaño se marcará primero en su extremo 5' con [γ-³²P]ATP y polinucleótido quinasa, y a continuación se formará el complejo molde-iniciador siguiendo procedimientos ya descritos (Mas et al. J Mol Bio\ 2002; 323: 181-197; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). El ensayo se inicia incubando la RT (a 20-30 nM en concentración de enzima activa) y el complejo molde-iniciador (20-30 nM) durante 10 min a 37 °C. A continuación se añade el sustrato (por ejemplo, AZT-trifosfato o uno de los diastereoisómeros del AZTTPaS) a una concentración suficientemente alta (>20 μM) como para permitir la adición completa de un nucleótido en el extremo 3' del iniciador. La reacción de rescate se lleva a cabo añadiendo posteriormente una mezcla de los cuatro dNTPs naturales (todos a concentración 100 μM, salvo el dATP que se suministra a 1 μM) y ATP (u otro ribonucleótido trifosfato) a 3.2 mM. La baja concentración de dATP es necesaria para evitar la formación de "dead-end complexes", tal como se describe con detalle en trabajos previos de nuestro grupo (Mas et al. EMBO J 2000; 19: 5752-5761; Mas et al. J Mol Bio\ 2002; 323: 181- 197; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). La reacción se detiene añadiendo 10 mM EDTA disuelta en formamida al 90 %. Cuando hay rescate, y por tanto escisión del inhibidor del extremo 3' terminal del iniciador, se permite que la síntesis de ADN continúe hasta alcanzar el final del molde, apareciendo productos largos, que se separan en geles desnaturalizantes de poliacrilamida al 20 % con urea 8 M. Los resultados se analizan utilizando placas fotoestimulables y un lector de placas (por ejemplo, BAS 1500 scanner, Fuji) con su correspondientes programas de análisis, para la cuantificación de los resultados.

It can be purchased from TriLink Biotechnologies (San Diego, California, USA), whose preparations contain the S _p diastereoisomer in a 2 to 1 ratio, with respect to R _v . Both shallow diastereoi can be separated by reverse phase chromatography (HPLC) using a Vydac C ₁₈ 218TP54 column (4.6 x 250 mm), and eluting under isocratic conditions (5% acetonitrile in 10 mM triethylammonium acetate buffer, pH 6.8). Under these conditions, elute the Sp diastereoisomer first and then the R _v . The triethylammonium cation can be exchanged with sodium cations by passing the solution through a Dowex 50WX8-200 cation exchange resin (Sigma). Incorporation assay and rescue can be carried out with a template-primer complex which mold the form a DNA oligonucleotide (for example, D38, ^{5'-GGGTCCTTTCTTACCTGCAAGAATGTATAGCCCTACCA-3 ')} and the initiator would be a shorter oligonucleotide, complementary to the previous one (for example, 25PG5A, 5 ^' -TGGTAGGGCTATACATTCTTGCAGG-3 ^' ) (Figure 2A). The smaller oligonucleotide will first be labeled at its 5 'end with [γ- ³² P] ATP and polynucleotide kinase, and then the template-primer complex will be formed following procedures already described (Mas et al. J Mol Bio \ 2002; 323 : 181-197; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). The assay is started by incubating the RT (at 20-30 nM in active enzyme concentration) and the template-initiator complex (20-30 nM) for 10 min at 37 ° C. The substrate (for example, AZT-triphosphate or one of the diastereoisomers of AZTTPaS) is then added at a sufficiently high concentration (> 20 μM) to allow the complete addition of a nucleotide at the 3 'end of the initiator. The rescue reaction is carried out by subsequently adding a mixture of the four natural dNTPs (all at 100 μM concentration, except for the dATP that is supplied at 1 μM) and ATP (or other ribonucleotide triphosphate) at 3.2 mM. The low concentration of dATP is necessary to avoid the formation of "dead-end complexes", as described in detail in previous work of our group (Mas et al. EMBO J 2000; 19: 5752-5761; Mas et al. J Mol Bio \ 2002; 323: 181-197; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). The reaction is stopped by adding 10 mM EDTA dissolved in 90% formamide. When there is rescue, and therefore excision of the inhibitor of the 3 'terminal end of the initiator, the DNA synthesis is allowed to continue until reaching the end of the mold, appearing long products, which are separated in denaturing gels of 20% polyacrylamide with urea 8 M. The results are analyzed using photostimulable plates and a plate reader (for example, BAS 1500 scanner, Fuji) with their corresponding analysis programs, for the quantification of the results.

The expression and purification of recombinant RTs used to demonstrate the concept justifying the invention has been described previously (Mas et al. EMBO J 2000; 19: 5752-5761; Mas et al. J Mol BM 2002; 323: 181-197 ; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). Using the above methodology, it is demonstrated that both in reactions catalyzed by wild RTs (for example, that of the BHlO strain), and by RTs resistant to multiple nucleoside-like inhibitors (for example, RT SS), both diastereoisomers of AZTTPaS (S _p and R _p ) can be incorporated into the DNA chain being synthesized. However, the incorporation of the S _p diastereoisomer is significantly more efficient (Figure 3A), which confirms previous observations obtained with the non-mutated RT and with the mutants D67N / K70R / T215F / K219Q and M41L / L210W / T215Y (Sluis-Cremer and Parniak. Antivir Ther 2004; 9: S29).

However, and surprisingly, once incorporated, none of the diastereoisomers can be removed from the 3 'terminal of the initiator by an RT with high phosphorolytic activity dependent on ATP as is the case of the SS SS (Figure 3B). This fact contrasts with what was observed with AZT, which in its triphosphate form is a good substrate for RT SS, but which once incorporated can be easily cleaved in the presence of high concentrations of ATP (Figure 2B). It is therefore demonstrated that the presence of a sulfur instead of oxygen as a substituent of the α phosphate of a nucleoside triphosphate, in which the ribose ring lacks a 3'-OH group, allows the incorporation of the nucleotide in the chain of DNA synthesized by RT, but prevents its elimination by ATP-dependent phosphorolysis. This result is extrapolated to phosphonates of formula (I) derived from acyclic nucleosides, such as tenofovir or adefovir, in which one of the sulfur-substituted phosphate substituents is replaced by sulfur.

The present invention further provides pharmaceutical compositions comprising a compound of formula (I) or (IB), or a pharmaceutically acceptable salt, derivative, prodrug, solvate or steroisomer thereof together with a pharmaceutically acceptable carrier, adjuvant, or carrier, for the administration to a patient

Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions) for oral, topical or parenteral administration.

In a preferred embodiment the pharmaceutical compositions are in oral form, either solid or liquid. Pharmaceutical forms suitable for oral administration they may be tablets, capsules, syrups or solutions and may contain conventional excipients known in the art, such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants for the preparation of tablets, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.

Solid oral compositions can be prepared by conventional methods of mixing, filling or tabletting. Repeated mixing operations can be used to distribute the active ingredient throughout all these compositions using large amounts of fillers. Such operations are conventional in the art. The tablets may be prepared, for example, by wet or dry granulation, and optionally coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in an appropriate unit dosage form. Suitable excipients, such as bulk agents, buffering agents or surfactants, may be used.

The aforementioned formulations will be prepared using usual methods such as those described or referred to in the Spanish and United States Pharmacopoeias and similar reference texts.

The administration of the compounds or compositions employed in the present invention can be by any suitable method, such as intravenous infusion, oral preparations and intraperitoneal and intravenous administration. Oral administration is preferred because of the comfort for the patient and the chronic nature of the diseases to be treated.

The compounds and compositions employed in this invention can be used with other drugs to provide a combination therapy. The other drugs may be part of the same composition or be provided as a separate composition for administration at the same time or at different times. The following examples are given only as an additional illustration of the invention, they should not be construed as limiting the invention as defined in the claims.

EXAMPLES Example 1.

Rescue of initiators terminated with AZT-monophosphate by ATP and adenosine-5'- γ-thiotriphosphate (ATPγS)

To TP

ATPγS

Rescue reactions of blocked initiators with nucleoside-like inhibitors were carried out using the D38 / 25PGA template-initiator complex (Figure 2A), under the conditions previously described (Mas et al. J Mol Biol 2002; 323: 181-197 ; Matamoros et al. J Biol Chem 2004; 279: 24569-24577). Briefly, the D38 / 25PGA complex (30 nM) was incubated for 10 min at 37 ° C, in the presence of the corresponding RT (20-30 nM, active enzyme concentration), in 25 μl of Hepes buffer pH 7.0 which it contained 15 mM NaCl, 15 raM magnesium acetate, 130 mM potassium acetate, 1 mM dithiothreitol (DTT) and 5% polyethylene glycol 6000 (weight / volume). The reactions were started by adding 25 μl of the same solution, in which we included AZT-triphosphate (obtained from Moravek Biochemicals, Brea, California, USA) at a final concentration of 25 μM. After incubating the samples for 30 min at 37 ° C, an elongated primer was obtained up to 26 nucleotides (see lanes B in Figure 2B).

The rescue reaction, which involves the cleavage of AZT-monophosphate and the subsequent elongation of the initiator, was carried out by adding a mixture of all dNTPs supplied at a final concentration of 100 μM (except dATP, which was supplied to 1 μM) and ATP 3.2 mM. After 2, 4, 6, 8, 10, 12, 15, 20 and 30 min 5 μl aliquots were collected which were mixed with 5 μl of a solution containing 10 mM EDTA in 90% formamide and 3 mg / ml xylene cyanol FF and 3 mg / ml Bromophenol Blue. The reaction products were resolved on a 20% polyacrylamide denaturing gel, containing 8 M urea, and the results were quantified by using photostimulable plates (BAS-IP MS 2040, Fujifilm), using a BAS 1500 scanner reader ( Fuji) and the Tina version 2.09 data analysis program (Raytest Isotopenmessgerate Gmbh, Staubenhardt, Germany). The percentage of primer (first) rescued is obtained from the intensity of the 38 nucleotide band, corresponding to the total elongation of the initiator, relative to the sum of this and that of 26 nucleotides.

Figure 2B shows that in the presence of ATP, the RT SS (carrier of the changes M41L / A62V / D67N / T69SSS / K70PJV118I / M184I / L210W / T215Y) has a high capacity to rescue AZT-terminated initiators, while said Activity is practically nil in the case of wild RT, derived from the BHlO strain of HIV-I. However, if one of the ATP γ phosphate substituents is replaced by sulfur, the rescue capacity presented by the RT SS decreases significantly (around 50%), as can be seen in Figure 2C.

These results demonstrate that substitutions that affect the oxygen involved in the rescue reaction can be determinants of their rescue efficacy.

Example 2

Rescue of blocked initiators as a result of the incorporation of 3'-azido-3'-deoxythymidine-5'-0- (l-thiotriphosphate) (AZTTPαS)

AZTTPaS Rescue reactions have been carried out in this case using the same procedure as in Example 1. Here the only difference lies in the use of AZTTPaS, instead of AZT-triphosphate in those experiments in which the incorporation of AZTTPaS (Figure 3A), or its rescue (Figure 3B). The S _P and R _P diastereoisomers of AZTTPaS, obtained after chromatographic separation in reverse phase HPLC, have been studied separately from the commercial product supplied by Trilink Biotechnologies. All rescues have been carried out in the presence of 3.2 mM ATP.

In the presence of a relatively high concentration of AZTTPaS (25 μM), RT SS is capable of incorporating both its diastereoisomer S _v and the diastereoisomer R _v , although the former is incorporated at a rate 12 times higher than the latter, manifesting itself as the best substrate of the polymerization reaction.

The analysis of the results of rescue reactions carried out with the RT SS showed that this enzyme is capable of effectively rescuing initiators blocked with AZT, but does not act (or acts very weakly) on blocked initiators after the incorporation of diastereoisomers of AZTTPaS. If the rescue kinetics of finished primers is compared with the incorporation product of the S _P diastereoisomer of AZTTPaS, with the results obtained in control tests carried out in the absence of ATP, there are practically no differences. In the case of the diastereoisomer R _v , a very low activity is observed when compared with its corresponding control.

These results show that α-phosphorothioates derived from AZT are not cleavable by the ATP-dependent phosphorolytic activity exhibited by many thymidine analog resistant enzymes.

Claims

1. The use of a compound of formula (I) or (IB)

(I) (B) where n is selected from O, 1 and 2; A is a C ₁ -C ₃ substituted or unsubstituted alkyl group, R is any of the purine or pyrimidine nitrogenous bases; Y R 'and R "are independently selected from H and a C ₁ -C ₁₀ alkyl group, R'" is hydrogen, an azide group or does not exist when there is a double bond between the 2 'and 3' carbons, the dotted line between carbons 2 'and 3' indicates the possibility of a double bond, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof, for the preparation of a medicament for the treatment of a patient infected with HIV and / or affected by AIDS. 2. Use according to claim 1 wherein R 'is hydrogen. 3. Use according to any one of claims 1 or 2 wherein R "is hydrogen. 4. Use according to any one of claims 1 to 3 wherein R '"is hydrogen or an azide group. 5. Use according to any one of claims 1 to 3 wherein in the compound of formula (I) A is the group -CH ₂ CH ₂ - or the group -CH (CH ₃ ) CH ₂ -. 6. Use according to any one of claims 1 to 4 wherein the compound of formula (IB) is 3'-azido-3'-deoxythymidine-5'-O- (l-thiophosphate) (AZTTPaS). 7. Use according to any of claims 1 to 4 wherein the compound is an analogue to any of the compounds zidovudine, stavudine, didanosine, lamivudine, zalcitabine, abacavir, emtricitabine, tenofovir, or adefovir, where oxygen has been replaced by sulfur as a substituent of α phosphate. 8. Use according to any one of claims 1 to 7 wherein the patient exhibits resistance to human immunodeficiency virus reverse transcriptase inhibitor drugs. 9. Use according to claim 8 wherein the patient exhibits resistance to any of the drugs zidovudine, stavudine, didanosine, lamivudine, zalcitabine, abacavir, emtricitabine, tenofovir, adefovir or mixtures thereof. 10. Pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or (IB) as defined in any of claims 1-7, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, and the less a carrier, adjuvant, or pharmaceutically acceptable carrier, for administration to a patient. 11. A compound of formula (I)

(I) where n is selected from 0, 1 and 2; A is a C ₁ -C ₃ substituted or unsubstituted alkyl group, R is any of the purine or pyrimidine nitrogenous bases; Y R 'and R "are independently selected from H and a C ₁ -C ₁ O alkyl group, or a pharmaceutically acceptable salt, a prodrug or a solvate thereof. 12. A compound according to claim 11 wherein R 'is hydrogen. 13. A compound according to any of claims 11 or 12 wherein R "is hydrogen. 14. A compound according to any of claims 11 to 13 wherein in the compound of formula (I) A is the group -CH ₂ CH ₂ - or the group -CH (CH ₃ ) CH ₂ -.