MXPA00013013A - ARYL PHOSPHATE DERIVATIVES OF d4T HAVING ANTI-HIV ACTIVITY - Google Patents

Abstract

Aryl phosphate nucleosise derivatives of formula (I) show potent activity against HIV without undesirable levels of cytotoxic activity. Examples of aryl phosphate nucleoside derivatives include aryl phosphate derivatives of d4T with para-bromo substitution on the aryl group. In particular, these derivatives are potent inhibitors of HIV reverse transcriptase. In a preferred aspect of the present invention, the phosphorus of the aryl phosphate group is further substituted with an amino acid residue that may be esterified or substituted, such as a methoxy alaninyl group. In said formula, Y is oxygen or sulfur;R1 is unsubstituted aryl or aryl substituted with an electron-withdrawing group;R2 is a nucleoside of formula (II or III).

Description

f ~ -, X 1 DERIVATIVES OF ARIL PHOSPHATE OF d4T THAT HAVE ANTI-HIV ACTIVITY Field of the Invention The present invention is directed to nucleoside derivatives, particularly aryl phosphate derivatives of 273'-didehydro-2 \ 3'-dideoxythymidine (hereinafter "d4T") which exhibits potent activity against the immunodeficiency virus. human (HIV), for example, as inhibitors of HIV reverse transcriptase. BACKGROUND OF THE INVENTION The dissemination of AIDS and the efforts to control the responsible virus are well documented. One way to control HIV is to inhibit its reverse transcriptase (RT) activity. This Thus, novel, potent and selective inhibitors of VI H RTs are useful as useful therapeutic agents. Potent known inhibitors of HIV RT include 5'-triphosphates of 2 ', 3'-deoxynucleoside analogues ("ddN"). These active RT inhibitors are generated intracellularly through the kinase action of Nucleoside and nucleotide kinase. In this way, these ddN compounds, such as AZT and d4T, have been considered to hold great promise in the search for anti-HIV agents. The limiting rate step for the conversion of 3'-asido-3'-deoxythymidine (Zidovudine; AZT) to its bioactive metabolite, AZT-25 triphosphate, appears to be the conversion of the monophosphate derivative to the diphosphate derivative, while the step of limiting regimen for the intracellular generation of the metabolite of 2 \ 3'-dideoxy-2 \ 3'-didehydrotimidine a (d4T), D4T-triphosphate, was reported as the conversion of the ptoseride to its monophosphate derivative (Balzarini et al., 1989 , J. Biol. Chem. 264: 6127; McGuigan et al., 1996, J. Med. Chem. 39: 1748). See Figure 1 for the mechanism proposed in the prior art. In an attempt to overcome the dependence of ddN analogs on intracellular nucleoside kinase activation, McGuigan et al. Prepared aryl methoxyalanilinphosphate derivatives AZT (McGuigan et al., 1993 J. Med. Chem. 36: 1048; McGuigan et al., 1992 Antiviral. Res. 17: 211) and d4T (McGuigan et al., 1996 J. Med. Chem. 39: 1748); McGuigan et al., 1996 Bioorg. Med. Chem. Lett. 6: 1 183). It has been shown that such compounds undergo intracellular hydrolysis to produce monophosphate derivatives which are further phosphorylated via thymidylate kinase to give the bioactive triphosphate derivatives in an independent form of thymidine kinase (TK). However, all attempts to update further improve the potency of the aryl phosphate derivatives of d4T through various substitutions of the aryl portion without concomitantly improving its cytotoxicity. (McGuigan et al., 1996 J, Med. Chem. 39: 1748). In the present invention, it has been discovered that a substitution in the aryl moiety in the aryl phosphate derivatives of nucleosides with an electron withdrawing portion, such as a para-bromo substitution, improves the ability of the d4T nucleoside derivatives to undergo hydrolysis due to the electron withdrawing property of the substituent. The phenyl substituted phosphate nucleoside derivatives demonstrate a potent specific antiviral activity.

SUMMARY OF THE INVENTION The present invention is directed to aryl phosphate nucleoside derivatives, particularly derivatives of 2'3'-didehydro-2 ', 3'-dideoxythymidine aryl phosphate (hereinafter referred to as "d4T"), which exhibits a potent activity against VI H, for example, as inhibitors of VI H reverse transcriptase. The aryl phosphate derivatives of d4T, for example, derivatives having an electron withdrawing substitution such as a para-bromo substitution in the aryl group, were unexpectedly found to exhibit markedly increased potency as anti-VI H agents without undesirable levels. of cytotoxic activity. In particular, these derivatives are potent inhibitors of HIV reverse transcriptase. In a preferred aspect of the present invention, the phosphorus of the aryl phosphate group is further substituted with an amino acid residue that can be esterified or substituted, such as a methoxy alaninyl group. For example, the d4T para-bromine-substituted phenylmethoxyandinphosphate derivative as an anti-HIV active agent, potently inhibits HIV replication in peripheral blood mononuclear cells (PBMNC) as well as in TK-deficient CEM T cells without any detectable toxicity . In addition, this novel derivative d4T, d4T-5 '- (para-bromophenyl methoxylaninyl phosphate), had a potent viral activity against RTMDR-1, a strain resistant to AZT and NNI of HIV-1, and moderate activity against HIV-2. Similarly, the phenylmethoxyalaninyl phosphate derivative substituted with para-bromine of AZT showed potent anti-HIV activity in PBMNC as well as TK-deficient CEM T cells, but was not effective against the RTMDR-1 resistant to AZT and NN I or VI H- 2. In contrast to these derivatives d4T and AZT the corresponding 3dT derivative, 3dT-5 '- (para-bromophenyl methoxylaninyl phosphate), while showing an improved activity on 3dT, was not as active as the derivatives of d4T and AZT in PBMNC or cells T CEM deficient TK. For knowledge, this is the first report of a relationship of structure activity previously not appreciated determining the potency of the phenyl phosphate derivatives of both d4T and AZT. The main compounds d4T-5 '- (para-bromophenyl methoxylaninyl phosphate) and AZT-5' - (para-bromophenyl methoxylaninyl phosphate) provide a basis for the design of effective HIV treatment strategies capable of inhibiting HIV replication, particularly in TK-deficient cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of a metabolic path proposed by the prior art for aryl phosphate derivatives of d4T. Figures 2A and 2B are diagrams showing the electron withdrawal hypothesis for the improved hydrolysis of a substituted phenyl ring. Figure 2C is an elusion profile showing production A-d4T as a result of hydrolysis of each of the compounds tested: Compound 2, wherein X = H (open squares); Compound 3, where X = OCH3 (filled squares); and Compound 4, where X = Br (full circles). Figure 2D is an elusion profile showing the sensitivity of the tested compounds to enzymatic hydrolysis via porcine liver esterase. Figure 3 is an elusion profile showing the intracellular hydrolysis of compounds 2-4 in TK-deficient CEM cells. A metabolite peak corresponding to 680 pmoles of A-d4T-MP was detected only in aliquots of lysates of CEM cells incubated with compound 4. Figures 4A-4F show the structures of compound 6c (Figure 4A) and compound 7c (Figure 4B); HIV activity against HTLVII IB in PBMNC and CEM T cells deficient in TK for compound 6c (Figure 4C) and for compound 7c (Figure 4D); and antiviral activity against VI H-1 (HTLVI II B), HIV-2 and RTMDR-1 for compound 6c (Figure 4E) and compound 7c (Figure 4F). Antiviral activity was expressed as% inhibition of HIV replication as measured by RT activity in infected cells. Figures 5A and 5B are schematic diagrams showing the effect of resonance (electron delocalization) on the phenyl ring, whereby the substituent for and the ortho substituent on the phenyl ring are expected to have the same electronic effect.

Detailed Description of the Invention It has been unexpectedly discovered that certain substituted aryl phosphate derivatives of nucleosides possess increased activity against HIV, while maintaining low levels of cytotoxicity. As such, these derivatives are particularly useful as active agents for antiviral compositions and for methods of treating viral infections such as VI H infections.

Compounds of the Invention The compounds of the invention as discussed more fully in the examples below are nucleoside aryl phosphate derivatives, particularly derivatives of d4T and AZT, which have potent viral activities. A nucleoside derivative suitable for use in compositions and methods of the inventions of the formula: or a pharmaceutically acceptable salt thereof, wherein Y is oxygen or sulfur, preferably oxygen; Ri is unsubstituted aryl or aryl substituted with an electron withdrawing group; R2 is a nucleoside of the formula II or II: (II) (III) wherein R6 is purine or pyrimidine, preferably pyrimidine, and R7, R1, R9. R10, R11 and R? 2 are independently hydrogen, hydroxy, halogen, azido, -NO2, -NR13R1- ", or -N (OR? 5) R? 6, wherein R13, R? , R15 and R16 are independently hydrogen, acyl, alkyl, or cycloalkyl; R3 is hydrogen or, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 or R4 can be taken together to form the side chain of proline or hydroxyproline; and R5 is hydrogen, alkyl, cycloalkyl, or aryl. The term "aryl" includes aromatic hydrocarbyl, such as, for example, phenyl, including fused aromatic rings such as for example, such group can be substituted or unsubstituted in the aromatic ring through a group of removal of electrons, such as, for example, halogen (bromine, chlorine, fluorine, iodine), NO2 or acyl. Preferably, the aryl substituted with an electron withdrawing group is bromophenyl, most preferably 4-bromophenyl. The term "acyl" includes substituents of the formula R17C (O) -where R? 7 is hydrogen, alkyl, or cycloalkyl. The term "alkyl" includes a straight or branched saturated aliphatic hydrocarbon chain having from 1 to 6 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl, tert-butyl (1 , 1-dimethylethyl), and the like. Said groups can be unsubstituted or substituted with hydroxy, halogen, azido, -NO2, -NR? 3R14, or -N (OR? 5) Ri6, wherein R13, R14, R15, and Rie are as defined above. The term "cycloalkyl" includes a saturated aliphatic hydrocarbon ring having from 3 to 7 carbon atoms, such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Said groups can be unsubstituted or unsubstituted with hydroxy, halogen, azido, -NO2, -NR13R14, or -N (OR? S) Ri6, wherein R13, R? 4, R15, and R16 are as defined previously. The term "purine" includes adenine and guanine. The term "pyrimidine" includes uracil, thymine, and cytosine.

Preferably the pyrimidine is thymine. The term "side chain of an amino acid" is the variable group of an amino acid and includes, for example, the side chain of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, hydroxylysine, isoleucine, ieucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, and the like. Preferably, the side chain of an amino acid is the side chain of alanine or tryptophan. In general, compounds substituted with an electron withdrawing group such as a halogen substituted with ortho or para or NO2 as shown in Figures 5A and 5B, provide more efficient hydrolysis for active inhibitor compounds. Substitution with halogen is preferred, and para-bromo substitution is very preferred. To generally illustrate the compositions and methods of the invention, derivatives of d4T are described. The d4T derivatives have aryl phosphate substitution, with the aryl group having an electron withdrawing substitution, such as an ortho or para substitution with a halogen (Br, Cl, F, I) or with a NO2 substitution. An example is shown below, wherein R 8 is an amino acid residue that can be esterified or substituted, for example, -NHCH (CH 3) COOCH 3 or pharmaceutically acceptable salts or esters thereof.

Synthesis of the d4T Derivatives: To synthesize the synthesis of the compounds of the invention generally, the synthesis of the d4T derivatives is described. The d4T derivatives can be prepared as follows. D4T can be prepared through thymidine through the methods described by Mansuri, et al., 1989, J. Med. Chem. 32, 461, the disclosure of which is incorporated herein by reference. Appropriately substituted arylphosphorochloridate can be prepared through the methods discussed by McGuigan, et al., Antiviral Res., 1992, 17:31 1, the disclosure of which is incorporated herein by reference. The phosphorocloridate is added to a solution of d4T in anhydrous THF containing N-methylimidazole to form the desired product. The d4T derivatives are administered to patients in the form of suitable compositions containing the d4T and AZT derivative as an active agent as a pharmaceutically acceptable auxiliary carrier or diluent. Sustained-release dosage forms may be used, if desired. The compositions are administered to a patient in need of antiviral activity in a suitable antiviral amount, for example, sufficient to inhibit HIV reverse transcriptase and / or inhibit VI H replication in host cells. The dose is administered according to an adequate dose regimen.

EXAMPLES The invention will now be explained with additional reference to the following examples, which should not be considered as limiting the invention.

EXAMPLE 1 Synthesis v Characterization of d4T Derivatives D4T 1 was prepared from thymidine following the procedure of Mansuri et al., 1989 J. Med. Chem. 32, 461. Properly substituted methoxylaninylphosphorochlorhydrates were also prepared with phenyl according to the reported method. by McGuigan et al., 1992 Antiviral Res., 17, 311. Compounds 2-4 were synthesized as presented in Scheme 1 below. 4T (l) Scheme 1. Phenylmethoxyalaninylphosphorochloridate was added to the solution of d4T and 1-methylimidazole in THF anhydrous and the mixture was stirred at room temperature for 5-6 hours. The process of the reaction mixture developed the required derivatives in good yields. Column chromatography was applied to obtain pure compounds. The physical data of the synthesized compounds were determined through HPLC which was conducted using a C18 4x250 mm LiChrospher column eluted with 70:30 water / acetonitrile at a flow rate of 1 ml / minutes. The purity of the following compounds exceeded 96% by H PLC. The 13 C NMR peaks marked by stars are divided due to diastereomers.

Compound 2: yield: 81%; IR (Net): 3222, 2985, 2954, 1743, 1693, 1593, 1491, 1456, 1213, 1 153, 1039, 931, 769 cm "1; 1 H NMR (CDCI3) d 9.30 (br s, 1H), 7.30-7.10 (m, 6H), 6.85-6.82 (m, 1H), 6.36-6.26 (m, 1H), 5.91-5.85 (m, 1H), 5.00 (br m, 1H), 4.19-3.68 (m, 4H), 3.72, 3.71 (s, 3H), 1.83, 1.80 (d, 3H), 1.38-1.25 (m, 3H), 13C NMR (CDCI3) d 173.9, 163.7, 150.7, 149.7, 135.7 *. 133.2 *. 129.6 *. 127.3 *. 125.0 *. 120.0, 111.1, 89.6 *, 84.5 * .66.9 *, 52.5 * .50.0 *, 20.9 and 12.3; 31P NMR (CDCI3) d 2.66, 3.20; mass MALDI-TOF m / e 487.9 (M + Na); HPLC retention time: 5.54 &5.85 minutes Compound 3: yield: 92%; IR (Net): 3223, 3072, 2999, 2953, 2837, 1743, 1693, 1506.1443, 1207, 1153, 1111, 1034, 937, 837 and 756 cm "1; 1 H NMR (CDCl 3) d 9.40 (br s, 1 H), 7.30-7.00 (m, 5 H), 6.83-6.81 (m, 1 H), 6.37-6.27 (m, 1 H), 5,915.86 (m, 1 H) , 5.00 (br m, 1H), 4.40-4.30 (m, 2H). 4.20-4.10 (m, 2H), 3.95-3.93 (s.3H), 3.82-3.80 (s.3H), 1.85-1.81 (s, 3H) and 1.39-1.29 (m.3H); 13 C NMR (CDCl 3) d 174.0, 163.9, 156.6, 150.8, 143.5, 135.8 *. 133.3 *, 127.4 *. 121.2 *, 114.5, 111.2, 89.7 *. 84.5, 66.9 * .55.5, 52.5, 50.6 *, 20.9, and 12.3; 31 i NMR (CDCI3) d 3.82, 3.20; MALDI-TOF mass m / e 518.2 (M + Na); HPLC retention time: 5.83 & 6.26 minutes Compound 4: yield: 83%; IR (Net): 3203, 3070, 2954, 2887, 2248, 1743, 1693. 1485, 1221. 1153. 1038, 912, 835, 733 cm "1; 1H NMR (CDCI3) d 9.60-9.58 (br s, 1H ), 7.45-7.42 (m, 2H), 7.30-7.09 (m, 4H), 6.37-6.27 (m, 1H), 5.93-5.88 (m, 1H), 5.04-5.01 (br m, 1H), 4.35- 4.33 (m, 2H) .4.27-3.98 (m, 2H), 3.71-3.70 (s, 3H), 1.85-1.81 (s, 3H), 1.37-1.31 (m, 3H); 3C NMR (CDCI3) d 173.7 , 163.8, 150.8, 149.7 *. 135.6 *. 133.1 *, 127.4 *. 121.9 *. 118.0, 111.2 *. 89.7 *. 84.4 *. 67. 8 *. 52.5, 50.0 *. 20.7. and 12.3; 31 P NMR (CDCl 3) d 3.41, 2.78; MALDI-TOF mass m / e 567.1 (M + Na); HPLC retention time: 12.04 & 12.72 minutes Example 2 Susceptibility of Compounds 2-4 for Hydrolysis Figures 2A and 2B show a schematic representation of the electronic effects of the substituent on the phenyl ring of the precursor of metabolite B (see Figure 1). To determine the susceptibility of the compounds for hydrolysis, compounds 2-4 were dissolved in methanol then treated with 0.0002 N NaOH. The concentrations were kept constant and the generation of the hydrolysis product A-d4T-MP was verified using HPLC. A Lichrospher column (C 18) was used for H PLC operations. The column was eluted under sodium conditions using the solvent 70:30 water / acetonitrile mixture and the elution profile is shown in Figure 2C. The hydrolysis of the compounds was tested in a porcine liver esterase system. The data is shown in Figure 2C. Compounds 2 and 4 (1 mM in Tris-HCl) were incubated with 100 U of porcine liver esterase (Sigma) in Tris-HCl pH buffer (pH 7.4) for 2 hours at 37 ° C. the reaction was stopped by adding acetone and cooling the reaction mixture. After centrifugation at 15,000 x g, 1.1 mL of aliquots of the reaction mixture were examined for the presence of the active metabolite, A-d4T-M P, using a quantitative analytical H P LC method capable of detecting 50 metabolite pinoles. The aliquot of 0. 1 mL of the reaction product of Compound 4 contained 0.4 nmoles of A-d4T-MP, while no metabolite was detected in the reaction product of Compound 2. As shown in Figures 2A and 2B , the presence of an electron withdrawing substituent in the para position of the phenyl portion probably increases the hydrolysis rates of the phenoxy group in the metabolite precursor B (Figure 2A and 2B) generated by the first step dependent on carboxiesterase (Figure 1 , A to B) of the metabolic path of d4T phenyl phosphate derivatives. A single substitution of bromine in the para position of the feni ring could not interfere with the recognition and hydrolysis of this compound through the carboxyesterase (Step A to B in Figure 1). An electronic effect induced by the para-bromo electron withdrawing substituent can result in an improved hydrolysis of the phenoxy C group producing D and subsequently E, the precursors of the key metabolite A-d4T-M P-In order to test this hypothesis, the unsubstituted compound 2, compound 3 substituted with para-methoxy (OCH3), and compound 4 substituted with para-bromine (= d4T-5 '- [p-bromo-phenylmethoxyalan inyl phosphate] or d4T-pBPMAP), for its rate of chemical hydrolysis after its treatment with 0.002 N NaOH by measuring the generation of alaninyl-d4T-monophosphate (A-d4T-MP). As shown in Figure 2C, compound 4 with a para-bromo substituent showed a much faster hydrolysis rate than unsubstituted compound 2, while compound 3 with the electron donation substituent -OCH3, in the position for, it had a slower hydrolysis rate than either of the two compounds. Similarly, the main compound 4 was more sensitive to the enzymatic hydrolysis via porcine liver esterase than the compound 2 (Figure 2D).

Example 3 Intracellular Metabolism of Compounds 2-4 in TK Cells Deficient in TK To analyze the intracellular metabolism of compounds 2-4 in TK-deficient cells, 1 × 10 6 CEM cells were incubated with compounds 2-4 (100 μM ) for 3 hours, and subsequently the formation of the partially hydrolyzed diester phosphate metabolite, alaninyl d4T monophosphate, was examined by HPLC. Notably, the amount of this metabolite in the CEM cells treated with compound 4 was substantially higher than in the CEM cells treated with compound 2 or 3 (680 pmoles / 106 cells vs. 50 pmoles / 106 cells; Figure 3). The CEM cells were cultured in a medium composed of RPMI, 10% fetal bovine serum, and 1% penicillin / streptomycin. Ten million cells at a density of 106 cells / L were incubated with 100 μM of this compound for 3 hours at 37 ° C. After incubation, the cells were washed twice with ice-cold PBS and extracted through the addition of 0.5 ml of 60% methanol. The cell lysates were kept at -20 ° C overnight, after which the lysates were centrifuged at 15,000 x g for 10 minutes to remove the cell waste. Aliquots of 100 μL of these lysates were injected directly into H PLC. The HPLC system consisted of a Hewlett Packard (H P) series 1 100 equipped with a quaternary pump, an autosampler, an electronic degasser, a diode array detector, and a computer with a chemical station software program for data analysis. The samples were eluted in a column of 250x4.6 mm Sulpelco LC-DBC18. A solvent gradient was used to resolve the metabolite of the parent compound, which consisted of a mixture of methanol and 10mM ammonium phosphate (pH 3.7). The gradient ran at a flow rate of 1 mL / minute from 5 to 35% methanol for the first 10 minutes, was maintained at 35% methanol for 5 minutes and ended with a linear gradient of 35 to 100% methanol in the next 20 minutes. The detection wavelength was set at 270 nm. A metabolite peak with a retention time of 8.47 minutes corresponding to 680 pmoles of A-d4T-MP was detected only in aliquots of lysates of CEM cells incubated with compound 4. Due to its improved susceptibility to hydrolysis, compound 4 it was postulated as a more potent anti-HIV agent than the other compounds. Compounds 2-4 as well as the compound of d4T (1) origin, were tested for their ability to inhibit the replication of VI H in peripheral blood mononuclear cells and TK deficient CEM T cells, using the previously described procedures, (Zarling , and others 1990 Nature 347: 92, Erice et al, 1993 Animicrob, Agents Chemoter, 37: 835, Uckun et al, 1998 Antimicrob, Agents Chemoter, 42: 383). The percentage of inhibition of viral replication was calculated by comparing the activity values of p24 and RT from the infected cells treated with the test substance with that of untreated infected cells. In parallel, the toxicity of the compounds was examined using a cell proliferation microculture tetrazolium (MTA) assay as described by Zarling, Enrice, and Uckun, Supra). The similarity of the IC50 values for the inhibition of VI H-1 replication, shown in Table 1, provides evidence that the derivatives of d4T-arylphosphate were not more potent than the compound of d4T origin when tested in cells mononuclear cells of peripheral blood infected with HIV-1. According to previous reports, the ability of d4T to inhibit HIV-1 replication was substantially reduced in TK-deficient CEM cells. While, the IC5o value of p24 production through d4T was 18 nM in peripheral blood mononuclear cells, it was 556 nM in TK-deficient CEM cells. Similarly, the ICso value to inhibit RT activity increased from 40 nM to 2355 nM (Table 1). Since the three aryl phosphate derivatives were more potent than d4T in TK-deficient CEM cells, compound 4 (d4T-5 '[p-bromo phenylmethoxyalaninylphosphate]) having a para-bromo substituent on the aryl portion was 1 2.6 sometimes more potent to inhibit the production of p24 (values of ICso'- 44 nM vs 556 nM) and 41.3 times more potent to inhibit RT activity (IC50 values: 57 nM vs 2355 nM) than d4T.

Table 1 None of the tested compounds exhibited any detectable cytotoxicity in peripheral blood mononuclear cells or CEM cells at concentrations as high as 10,000 nM, as determined by MTA. Integrally, compound 3 with a para-methoxy substituent in the aryl portion was 5.6 times less effective than compound 4 in inhibiting RT activity in TK cells deficient in TK infected by VI H (IC5o values: 320 nM vs 57 nM) although these two compounds showed similar activity in peripheral blood mononuclear cells (IC 50 values: 33 nM vs 42 nM). Thus, the identity of the substituent for seems to affect the anti-HIV activity of d4T aryl phosphate derivatives in TK deficient cells. For the knowledge, this is the first demonstration that the potency as well as the selectivity index of d4T-aryl-phosphate derivatives can be substantially improved by introducing a single para-bromo substituent in the aryl portion. This previously unknown structure-activity relationship determined by the aryl portion of the d4T derivatives provides a basis for the design of potentially more potent d4T analogues.

Example 4 Activity of Compound 4 v AZT in MDR cells The activity of Compound 4 (d4T-5 '- [p-bromophenyl methoxyalaninyl phosphate]) against HIV-infected MDR cells was compared with AZT-5' - [p-bromophenyl methoxyalaninyl phosphate] (p-AZT) and with AZT. The incubation and analysis methods used were described above for Example 4. As shown in Table 2, P-AZR and AZT have similar activities with the IC50 values of 1.5 and 2.0 nM, respectively. The activity of compound 4 (0.02 nM) is 100 times more effective than AZT (2.0 nM).

Table 2 Example 5 Synthesis of 3dT Aryl Phosphate Derivatives By way of further comparison, the effect on the anti-VI H activity of various substitutions on the aryl group of 3'-deoxythymidine aryl phosphate derivatives (3dT), was studied. As shown in Scheme 2, 3dt 5 was prepared from d4T1, which was prepared from thymidine using the literature method (Mansuri, et al. 1989 J. Med. Chem. 32: 461-466). ). Hydrogenation of 1 in ethanol in the presence of H2 and the catalytic amount of 5% Pd / C was performed to provide 3dT 5 in a yield of 85%. Phenyl substituted methoxylaninylphosphorochloridates were also prepared in an appropriate manner, according to the method reported by McGuigan et al., 1992 Antiviral Res 17:31 1 -321, and compounds 6-1 1 were synthesized as shown in Scheme 2. 3dT (5) 6: X = H 7: X = Cl 8: X = F 9: X = Br 10: X = NO-, ll: X = OCH3 Scheme 2. Methoxylaninylphosphorochloridate substituted with phenyl in appropriate form was added to a mixture of 3dT and 1-methylimidazole in anhydrous THF. The reaction mixture was stirred for 12 hours at room temperature and then the solvent was removed. The resulting gum was redissolved in chloroform and washed with 1 M HC1, saturated sodium bicarbonate solution (except in the case of the NO2 derivative) and then with water. The organic phase was dried through MgSO 4 and the solvent was removed in vacuo. The crude product was purified through silica gel evaporation column chromatography eluted using 5% methanol in chloroform to give the pure compounds in 6-11 in good yields. The physical data of the synthesized compounds were determined. HPLC was conducted using a C184x250 mm LiChrospher column eluted with 70:30 water / acetonitrile at a rate of 1ml / minute. The purity of the following compounds exceeded 96% through HPLC. The 13 C NMR peaks marked with stars were divided due to the diastereomers. Compound 5: yield: 85%; 1H NMR (CDC13) d 11.1 (br s, 1H), 7.82 (s, 1H), 5.97-5.94 (m, 1H); 5.10 (br s, 1H); 4.05-3.95 (m, 1H), 3.72-3.52 (m, 2H), 2.30-1.86 (m, 4H), 1.77 (s, 3H); 13C NMR (CDCI3) d 163.9, 150.4, 136.4, 108.7, 84.8, 81.4, 62.2, 31.8, 25.1, and 12. 5. Compound 6: yield: 96%; IR (Net): 3211, 2955, 2821, 1689, 1491, 1265, 1211, 1153, 1043 and 933 cm "1; 1H NMR (CDCI3) d 10.1 (br s, 1H), 7.47 (s, 1H), 7.32 -7.12 (m, 5H), 6.14-6.08 (m, 1H), 4.41-4.21 (m, 4H), 4.05-4.00 (m, 1H), 3.70, 3.69 (s, 3H), 2.37-2.32 (m, 1H) 2.05-1.89 (m, 7H), 1.38-1.35 (dd, 3H); 13C NMR (CDCI3) d 173.6 *, 163.8, 150.3, 150.1 *. 135.2, 129.4 *. 124.7, 119.8 *. 110.5 *. 85.7 *. 78.3 *, 67.2 * .52.3, 50.1 *, 31.6 * .25.4 * .20.7 * and 12.4 *; 31P NMR (CDCI3) d 2.82 &3.11; MS (MALDI-TOF): 490.4 (M + Na ); HPLC retention time = 6.86, 7.35 minutes Compound 7: yield: 96%; IR (Net): 3217, 2954, 2821, 1743, 1689, 1489, 1265, 1217, 1153, 1092, 1012, 926 & 837 cm "1; 1 H NMR (CDCl 3) d 9.40 (br s, 1 H), 7.43-7.41 (m, 1 H), 7.30-7.14 (m, 4 H), 6.13-6.07 (m, 1 H), 4.39-4.00 (m, 5 H), 3.71.3.70 (s.3H), 2.38-2.36 (m, 2H), 2.09-1.89 (m, 5H), 1.39-1.36 (dd, 3H); 13C NMR (CDCI3) d 173.6 *, 163.7, 150.2, 148.8 *, 135.3, 129.5-129.0, 121.5-121.3, 116.3, 110.6, 86.0 *. 78.4 *. 67.7 *. 52.6 *. 50.2 *. 31.8 *. 25.4 *. 20.9 * and 12.5; 31P NMR (CDCI3) d 2.87 & 3.09; MS (MALDI-TOF): 524.9 (M + Na); HPLC retention time = 14.05, 14.89 minutes. Compound 8: Viscous oil, yield: 96%; ? ma: 223 (e 3338) and 269 (e 4695) nm; IR (Net): 3211, 2955, 1743. 1693, 1500. 1569, 1265, 1197, 1153, 1045, 923 & 843 cm "1; 1 H NMR (CDCl 3) d 9.40 (br s, 1 H), 7.45-7.43 (d, 1 H), 7.19-7.01 (m, 4 H), 6.14-6.06 (m, 1 H), 4.39-3.97 ( m, 5H), 3.71, 3.70 (s, 3H), 2.38-1.89 (m, 7H), 1.39-1.35 (t, 3H); 13C NMR (CDCI3) d 173.6 *. 163.7, 150.2, 150.1 *, 135.3, 121.5 *. 116.3 *. 110.6 *. 85.9 *, 78.4 *. 67.7 *, 52.6, 50.2 *. 31.8 *, 25.6 *, 20.9 *, and 12.5; 31P NMR (CDCI3) d 3.13 &3.37; MS (MALDI-). TOF): 508.2 (M + Na); HPLC retention time = 8.38, 8.80 minutes Compound 9: yield: 83%; IR (Net): 3211, 295.4, 1743, 1689, 1485, 1265, 1217, 1153, 1010 , 923 &833 cm "1; 1 H NMR (CDCl 3) d 9.82 (br s, 1 H), 7.45-7.41 (m, 3 H), 7.15-7.11 (m, 2 H), 6.14-6.06 (m, 1 H), 4.39-4.00 (m, 5 H), 3.71, 3.70 (s, 3H). 2.38-1.89 (m, 7H), 1.39-1.35 (dd, 3H); 13C NMR (CDCl 3) d 173.6 *. 163.8, 150.3, 148.5 *, 135.2, 132.6 *. 121.8 *, 117.7, 110.6 * .85.9 * .78.3 *, 67.2 * .52.5, 50.2 *. 31.6 *, 25.6 *. 20.8 *. and 12.5; 31P NMR (CDCI3) 8 2.83 & 3..05; MS (MALDI-TOF): 570.0 (M + 2 + Na); HPLC retention time = 15.50, 16.57 minutes.

Compound 10: yield, 87%; IR (Net): 3203, 2955, 1743, 1684, 1593, 1522, 1348, 1265, 1153, 1101, 920 & 860 cm "; 1 H NMR (CDCl 3) d 9.51 (br s, 1H), 8.248.21 (m, 2H), 7.42-7.37 (m, 3H), 6.13-6.08 (m.H.), 4.39-4.03 ( m.5H), 3.72, 3.71 (s 3H), 2.38-1.89 (m.7H), 1.41-1.38 (dd, 3H), 13C NMR (CDCI3) d 173: 4 *, 163.7, 155.2 *. 150.2, 144.4, 135.3, 125.9-125.4, 120.6 *, 115.4, 110.6 * .86.1 * .78.4 *. 68.1 * .52.7, 50.2 * .31.7 * .25.8 * .20.9 * and 12.5; 31P NMR (CDCI3) d 2.60 & 2.81; MS (MALDI-TOF): 535.0 (M + Na); HPLC retention time = 8.12, 10.14 minutes Compound 11: performance, 100%; IR (Net): 3209, 2954, 1743, 1506, 1468, 1265, 1207, 1153, 1036, 937 & 835 cm "1; 1 H NMR (CDCl 3) d 9.89 (br s, 1H), 7.497.47 (m, 1H), 7.16-7.11 (m, 2H), 6.84-6.80 (m, 2H), 6.15-6.09 ( m, 1H), 4.39-4.02 (m, 5H), 3.77, 3.76 (s, 3H), 3.74, 3.73 (s, 3H), 2.38-1.89 (m, 7H), 1.38-1.33 (t, 3H); 13C NMR (CDCI3) d 173.7 *, 163.9, 156.3, 150.3, 143.7 *. 135.2, 120.7 *. 114.3 *. 110.5, 35.7 *. 78.4 *, 67.3 *. 55.4, 52.4, 50.1 *, 31.8 * .25.4 *, 20.8 * and 12.4 *; 31P NMR (CDCI3) d 3.27 &3.52; MS (MALDI-TOF): 521.3 (M + 1 + Na); HPLC retention time = 7.15, 7.66 minutes.

Example 6 Antiviral Activity of Compounds 6-11 of 3dT Compounds 6-11 as well as the compound of 3dT origin in tub were compared with d4T for their ability to inhibit replication of HIV-1 in peripheral blood mononuclear cells. and CK T cells deficient in TK using previously described methods (Zarling et al., 1990; Erice et al., 1993; Uckun et al., 1998, Supra). The 3dT as well as its derivatives were less active than d4T in peripheral blood mononuclear cells, as well as in TK T cells deficient in TK (Table 3). Notably, in peripheral blood mononuclear cells, the IC50 values [RT] for compounds 6-11 1 were greater than the IC50 value [RT] of 3dT (1.2-3.1 versus 0.7, Table 3), suggesting that these prodrugs they are sufficiently stable and the independent steps of TK in their metabolism, perhaps their enzymatic hydrolysis, can be of limiting regime for the generation of active species. In contrast, d4T aryl phosphate derivatives are reported to be more potent than d4T, suggesting that the TK-dependent generation of d4T monophosphate is limiting in its metabolic activation (McGuigan et al., 1996a). According to the results reported in the literature with respect to the biological activity of the aryl phosphate derivatives of d4T and AZT (McGuigan et al., 1993, 1993a), aryl phosphate derivatives of 3dT were more active than the compound of origin 3dT to inhibit replication of HIV-1 in TK-deficient cells, always with IC50 values [RT] of high micromolar value. Since compounds 6-1 1 were less active in TK-deficient CEM T cells than in peripheral blood mononuclear cells (PBMNC), it was postulated that the conversion of 3dT monophosphate generated from these prodrugs into their active triphosphate, In contrast, the d4T aryl phosphate derivatives showed similar activity in normal and TK-deficient cells (McGuigan et al, 1996 Bioorg, Med.Chem.Let.t. 6: 1183- 1186).

Anti-HIV activity of 3'-deoxythymidine aryl phosphate derivatives (6-11) in normal peripheral blood mononuclear cells (PBMNC) and TK-deficient CEM T cells.

All data are in μM and represent concentrations required to inhibit viral replication, as measured by RT activity assays, by 50% (IC50 [RT]) 9 or 50% cytotoxic concentration, as measured by MTA (IC5o [MTA]) (Mansuri et al., 1989, J. Med. Chem. 32: 461).

Table 3 As shown in Figures 5A and 5B, the electronic effect of the substitutions for in the phenyl ring should affect the hydrolytic conversion of B to D in the metabolic path of aryl phosphate derivatives of 3dT depicted in Figure 1. The presence of an electron withdrawing substituent in the para position of the phenyl portion was expected to increase the hydrolysis rates of the substituted phenoxy groups in compounds 7-10 (Figure 2A and 2B). However, these compounds were no more active than Compound 6 without substitution for or compound 1 1 with a substituent for electron donation, accelerating the hypothesis that the first step of carboxylesterase-dependent hydrolysis in their metabolism (A to B in Figure 1) plays a critical role and limiting regime for the generation of active 3dT metabolites. In this manner, compounds 7-10 can serve as relatively poor substrates for putative sensitive carboxyesterase for hydrolysis in accordance with the proposed metabolic pathway for arylmethoxyalaninylphosphate derivatives of nucleoside analogues (Mclntee et al., 1997 J. Med. Chem. : 3323-3331).

Example 7 Anti-HIV Activity of the d4T Derivatives. AZT. and 3dT As shown in Scheme 1, d4T 1 was prepared from thymidine using the literature procedure (Mansuri et al., 1989, Supra). Hydrogenation of 1 in ethanol in the presence of H2 and a catalytic amount of 5% Pd / C provided 3dT 3 in an 85% yield (Scheme 1). AZT 2 was prepared from thymidine using literature methods (C hu et al., US Patent No. 4,841,039). DdN phosphorylating agents possessing different substituents on their phenoxy portions 5a, 5b and 5c, were prepared from the phenols commercially available in two step procedures (Scheme 2) (McGuigan et al., 1992, Supra), in where compounds 4a, 4b, 5a, 5b, 7a and 7b were previously reported. Compounds 4c and 5c are novel and their synthetic procedures as well as their characterization data are reported below. The synthesis of phenylmethoxyalaninylphosphate derivatives of d4T 1, AZT 2 or 3dT 3 was performed following the condition of the literature as shown in Scheme 3. (McGuigan et al., 1992). The general synthetic procedures are as follows: methoxylaninylphosphorochloridate appropriately substituted with phenyl. 5, was added to a mixture of the desired ddN (1, 2 or 3) and 1-methylimidazole in anhydrous THF. The reaction mixture was stirred for 12 hours at room temperature and then the solvent was removed. The resulting gum was redissolved in chloroform and washed with 1 M HCl, a saturated sodium bicarbonate solution and then with water. The organic phase was dried through MgSO 4 and the solvent was removed in vacuo. The crude product was purified by flash column chromatography on silica gel using a solvent mixture of methanol and chloroform for elution to give the desired pure compounds in good yields.

Scheme 1 Synthesis of d4T and 3dT.

Scheme 2. Synthesis of phenyl Methoxylaninylphosphorochloridate.

Scheme 3. Synthesis of ddN phenylmethoxyalaninylphosphate derivatives.

P-Bromophenyl 4-phosphorodichloridate. Following the procedure described by McGuigan et al., 1993, Supra, a solution of p-bromophenol (13.20 g; 76.30 mmol) and 10.65 mL of distilled triethylamine in 165 mL of ET2O, was added dropwise in a vigorously stirred chloride solution. of phosphoryl (8.5 mL; 91.2 mmole) in 83 mL of anhydrous ET2O at 0 ° C for a period of 3 hours under a nitrogen atmosphere. Subsequently, the resulting mixture was gradually warmed to room temperature, stirred efficiently overnight at room temperature and then heated to reflux for 2 hours. The reaction mixture was cooled to room temperature and filtered under suction pressure. The precipitate was washed with anhydrous ET2O (2x50 mL). The combined ET2O layers were evaporated to dryness in a rotary evaporator to produce crude 4c as a pale yellow oil, which was then subjected to vacuum distillation to give pure 4c (14.05 g, 63.5% yield) as a colorless viscous oil. (eg 1 10- 1 15 ° C / 2 mm Hg). I R (Net) 3095, 1481, 1303, 1 187, 948, 829 - 1 cm 1 H NMR (300 MHz, CDCl 3) d 7.50 (2H, d, J = 9.0 Hz), 7.15 (2H, d, J = 9.0 Hz). GC / MS (m / e) 290 (M +), 254 (M + - Cl), 173 (M + - POCI2, 81 Br), 171 (M + - POCI 2, 79 Br), 156 (M + - PO 2 Cl 2, 81 Br), 154 (M + -PO2Cl2, 79 Br).

P-Bromophenyl methoxylaninylphosphorocloride 5c. Following the procedure described by McGuigan et al., Supra, a solution of distilled triethylamine (8.80 mL, 63.14 nmol) in 180 mL of anhydrous CH2Cl2 was added dropwise through an addition funnel to a vigorously stirred phosphorodichloridate solution of p-bromophenyl 4c (8.69 g, 29.97 mmol) and L-alanine methyl ester hydrochloride (4.19 g, 30.02 mmol) in 250 mL of anhydrous CH 2 Cl 2 at -70 ° C for a period of three hours under a nitrogen atmosphere. Subsequently, the resulting mixture was allowed to warm gradually to room temperature and was stirred overnight at room temperature. The solvent was stirred in a rotary evaporator. Anhydrous 300 mL ET2O was added to dissolve the residue and then filtered under suction pressure to remove the white solid. The white solid was rinsed with anhydrous ET2O (2x60 mL). The ET2O layers were combined and evaporated to dryness to give a quantitative yield of 5c (10.7 g) as a pale pink-yellow viscous oil. This product was then used for the next reaction step without further purification. IR (Net) 3212, 2989, 2952, 1747, 1483, 1270, 1209, 1 147, 927, 831, 757cm "1. 1 H NMR (300 MHz, CDCl 3) d 8.70 (1 H, br, Ala-N H ), 7.48 (2H, d, J = 9.0Hz, aril H), 7.16 (2H, d, J = 9.0Hz, aril H), 3.79 &3.77 (3H, s & s, -OCH3), 1.51 &; 1.40 (3H, d & d, Ala-CH3), MS (Cl, m / e) 357.9 (M +, 81 Br), 355.9 (M +, 79 Br), 322.0 (M + -Cl, 81 Br), 320.0 ( M + -Cl, 79Br), 297.9 (M + -COOCH3, 81 Br), 295.9 (M + -COOCH3, 7 Br), 184.0 (M + -BrC6H4O).

Characterization data of phenyl methoxyalaninylphosphate derivatives of AZT 1, d4T and 3dT 3: H PLC was conducted using a C18 4x250 mm LiChrospher column eluted with 70:30 water / acetonitrile at the flow rate of 1 ml / ml nuto The purity of the following compounds exceeded 96% through H PLC. The 13 NMR peaks marked with asterisks were divided due to the diastereomers that arise from the stereocenters of phosphorus. Compound 6a: yield: 81%; IR (Net): 3222, 2985, 2954, 1743, 1693, 1593, 1491, 1456, 1213, 1153, 1039, 931, 769 cm "1; 1 H NMR (CDCI3) d 9.30 (br s, 1H), 7.307.10 (m, 6H), 6.85-6.82 (m, 1H), 6.36-6.26 (m, 1H), 5.91-5.85 (m, 1H), 5.00 (br m, 1H), 4.19-3.68 (m, 4H), 3.72, 3.71 (s, 3H), 1.83, 1.80 (d, 3H), 1.38-1.25 (m, 3H), 13C NMR (CDCI3) d 173.9, 163.7, 150.7, 149.7, 135.7 *, 133.2 *. 129.6 *. 127.3 *. 125.0 *. 120.0, 111.1, 89.6 * .84.5 * .66.9 *, 52.5 *, 50.0 * .20.9 and 12.3; 31P NMR (CDCI3) d 2.66, 3.20; mass MALDI-TOF m / e 487.9 (M + Na); HPLC retention time: 5.54 &5.85 minutes Compound 6b: yield: 92%; IR (Net): 3223, 3072, 2999, 2953, 2837, 1743, 1693, 1506,1443, 1207, 1153, 1111, 1034, 937, 837 and 756 cm "1; 1 H NMR (CDCl 3) d 9.40 (br s, 1H), 7.30-7.00 (m, 5H), 6.83-6.81 (m, 1H), 6.37-6.27 (m, 1H), 5.91-5.86 (m, 1H), 5.00 (br m, 1H), 4.40-4.30 (m, 2H), 4.20-4.10 (m, 2H), 3.95-3.93 (s, 3H), 3.82-3.80 (s, 3H), 1.85-1.81 (s, 3H) and 1.39-1.29 (m, 3H); 13C NMR (CDCI3) d 174.0. 163.9, 156.6, 150.8, 143.5, 135.8 *. 133.3 *. 127.4 *. 121.2 *. 114.5, 111.2, 89.7 *. 84.5, 66.9 *. 55.5, 52.5, 50.6 *, 20.9, and 12.3; 31P NMR (CDCI3) d 3.82, 3.20; MALDI-TOF mass m / e 518.2 (M + Na); HPLC retention time: 5.83 & 6.26 minutes Compound 6c: yield: 83%; IR (Net): 3203, 3070, 2954, 2887, 2248, 1743, 1693, 1485, 1221, 1153, 1038, 912, 835, 733 cm "1; 1H NMR (CDCI3) d 9.60-9.58 (br s, 1H ), 7.45-7.42 (m, 2H), 7.30-7.09 (m, 4H), 6.37-6.27 (m, 1H), 5.93-5.88 (m, 1H), 5.04-5.01 (br m, 1H), 4.35- 4.33 (m, 2H), 4.27-3.98 (m, 2H), 3.71-3.70 (s, 3H), 1.85-1.81 (s, 3H), 1.37-1.31 (m, 3H); 13C NMR (CDCI3) d 173.7 , 163.8, 150.8, 149.7 *. 135.6 *. 133.1 *. 127.4 *. 121.9 *. 118.0. 111.2 *. 89.7 *. 84.4 *. 67. 8 *, 52.5, 50.0 *, 20.7, and 12.3; 31P NMR (CDCI3) d 3.41, 2.78; MALDI-TOF mass m / e 567.1 (M + Na); HPLC retention time: 12.04 & 12.72 minutes Compound 7c: yield: 95%; IR (Net) 3205.7, 3066.3, 2954.5. 2109.8, 1745.3, 1691.3, 1484.9, 1270.9, 1153.2, 1010.5 and 926.1 cm "1 H NMR (300 MHz, CDCl 3) d 8.69 (1H, br, 3-NH), 7.45 (2H, d, J = 9.0Hz, aryl H), 7.34 &7.32 (1H, s & , vinyl H), 7.11 (2H, d, J = 9.0Hz, aril H), 6.18 &6.13 (1H, t & t, J = 6.6 &6.6Hz, H in C1 '), 4.44-3.77 (6H, m, H in C-3', 4 '&5", Ala-NH and Ala-CH), 3.73 & 3.72 (3H, s &s, -COOCH3), 2.51-2.20 (2H, m, H in C-2 '), 2.18 (3H, s, -CH3 in C-5), 1.39 & 1.36 (3H, d & d, Ala-CH3). 13 C NMR (75 MHz, CDCl 3) d 173.6, 163.6, 150.1, 149.2, 149.1, 135.2, 132.4, 121.6, 117.8, 111.1. 85.0. 84.7. 81.9, 81.8, 65.5, 60.1, 59.9, 52.4, 50.0, 49.9, 36.9, 20.6, 20.5, 12.2. MS (Cl, m / e) 589.1 (M +, 81 Br) and 587.1 (M +, 79 Br). Compound 8a. yield: 96%; IR (Net): 3211, 2955, 2821, 1689, 1491, 1265, 1211, 1153, 1043 and 933 cm "1; 1H NMR (CDCI3) d 10.1 (br s, 1H), 7.47 (s, 1H), 7.32 -7.12 (m, 5H), 6.14-6.08 (m, 1H), 4.41-4.21 (m, 4H), 4.05-4.00 (m, 1 H), 3.70, 3.69 (s, 3H) 2.37-2.32 (rn, 1H), 2.05-1.89 (m, 7H), 1.38-1.35 (dd, 3H), 13C NMR (CDCI3) d 173.6 *. 163.8, 150.3, 150.1 *. 135.2, 129.4 *. 124.7, 119.8 *. 110.5 *. 85.7 * .78.3 * .67.2 * .52.3, 50.1 *, 31.6 * .25.4 *, 20.7 *. And 12.4 *; 31P NMR (CDCI3) d 2.82 &3.11; MS (MALDI-TOF): 490.4 (M + Na ); HPLC retention time = 6.86, 7.35 minutes.

Compound 8b: yield, 100%; IR (Net). 3209, 2954, 1743, 1506, 1468, 1265, 1207, 1153, 1036, 937 & 835 cm "1; 1 H NMR (CDCl 3) d S.89 (br s, 1 H), 7.49-7.47 (m, 1 H), 7.16-7.11 (m, 2 H), 6.84-6.80 (m, 2 H), 6.15- 6.09 (m.1H), 4.39-4.02 (m, 5H), 3.77, 3.76 (s, 3H), 3.74, 3.73 (s, 3H), 2.38-1.89 (m, 7H), 1.38-1.33 (t, 3H) ); 13C NMR (CDCI3) d 173.7 *. 163.9, 156.3, 150.3, 143.7 *. 135.2, 120.7 *, 114.3 *. 110.5, 85.7 *. 78.4 *. 67.3 *. 55.4, 52.4, 50.1 *., 31.8 * .25.4 * .20.8 * and 12.4 *; 31P NMR (CDCI3) d 3.27 &3.52; MS (MALDI-TOF): 521.3 (M + 1 + Na); HPLC retention time = 7.15.7.66 minutes Compound 8c: yield: 83%; IR (Net): 3211, 2954, 1743, 1689, 1485, 1265, 1217, 1153, 1010, 923 & 833 cm "1; 1 H NMR (CDCl 3) d 9.82 (br s, 1H), 7.45-7.41 (m, 3H), 7.15-7.11 (m, 2H), 6.14-6.06 (m, 1H), 4.39-4.00 ( m, 5H), 3.71, 3.70 (s, 3H), 2.38 1.89 (m, 7H), 1.39-1.35 (dd, 3H); 13C NMR (CDCI3) d 173.6 *, 163.8, 150.3, 148.5 *. 135.2, 132.6 * 121.8 *. 117.7, 110.6 * .85.9 * .78.3 *, 67.2 * .52.5, 50.2 * .31.6 * .25.6 * .20.8 *. And 12.5; 31P NMR (CDCI3) d 2.83 &3.05; MS (MALDI) -TOF): 570.0 (M + 2 + Na); HPLC retention time = 15.50, 16.57 minutes.

Cellular Assays of Anti-HIV Activity and Cytotoxicity. Anti-HIV activities were evaluated in peripheral blood mononuclear cells (PBMNC) infected with HIV-1 sensitive to AZT (strain: HTLVIIIB), infected by HIV-1 resistant to AZT and NNI (strain: RTMDR-1) - (cordially provided by Dr. Brendan Larder, NIH AI DS Research and Reference Reagent Program, DIV, AI DS, N IAI D, NI H; cat. # 2529), or infected with HIV-2 (strain: CBL-20), as well as CI? M T cells deficient in TK infected by HTLVI IIB determining the composition of the compound necessary to inhibit viral replication by 50%, based on reverse transcriptase assays (IC50 [RT]). The percentage of viral inhibition was calculated by comparing the RT activity values of the infected cells treated with the test substance with RT values of untreated infected cells (i.e., virus controls). 50% of the cytotoxic concentrations of the compounds (CCso [MTA]) was measured through the micro-culture tetrazolium assay (MTA), using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) hydroxide ) -5 - [(phenylamino) -céirbonyl] -2H-tetrazolium (XTT) (Zarling et al., 1990, Erice, 1993, Uckun et al., 1998, Supra).

Identification d4T-5 '(para-bromophenyl methoxylaninyl phosphate) and AZT-5' - (para-bromophenyl methoxylaninyl phosphate) as potent anti-HIV agents. The d4T phenylphosphate derivatives were not more potent than the compound of d4T origin when tested in PBMNCs infected with VI H-1. The ability of d4T to inhibit replication of VI H-1 was substantially reduced in TK-deficient CEM cells. While the IC50 value for the inhibition of RT activity through d4T was 40 nM in PBMNC, was 2400 nM in TK cells deficient in TK (Table 4 and Figures 4A-4F). Since the three phenyl phosphate derivatives were more potent than d4T in CE cells; TK deficient, compound 6c (d4T-5 '- [p-bromo phenylmethoxyalaninyl phosphate]) with a para-bromo substituent on the phenyl portion was 60 times more potent to inhibit RT activity (IC5o values: 60 nM vs 2400 nM) than d4T (Table 4). None of the compounds exhibited any detectable cytotoxicity for PBMNC or CEM cells at concentrations as high as 10,000 nM, as determined through MTA. Intriguingly, compound 6b with a para-methoxy substituent on the phenyl portion was 5 times less effective than compound 6c in inhibiting RT activity in TK cells deficient in TK infected by VI H (IC50 values: 300 nM vs. 60 nM), although these two compounds showed similar activity in peripheral blood mononuclear cells (IC5o values: 30 nM vs 40 nM) (Table 4). Compounds 7a, 7b, 7c and their compound of AZT 2 origin were tested for their ability to inhibit HIV replication in PBMNC and TK deficient CEM T cells (Table 4). The percentage of inhibition of viral replication was calculated by comparing the RT activity values of the infected cells treated with the test substance with those of untreated infected cells. In parallel, the cytotoxicity of the compounds was examined using a tetrazolium microculture (MTA) assay of cell proliferation. The ability of AZT 2 to inhibit the replication of VI H-1 was substantially reduced in TK-deficient CEM cells.

While the IC50 value for the inhibition of RT activity through AZT was 3 nM in PBMNC, it was 200 nM in TK-deficient CEM cells. In contrast to the corresponding d4T derivatives, the unsubstituted and unsubstituted phenyl phosphate derivatives of AZT were not more potent than the compound of AZT origin when tested in TK cells deficient in TK deficient in HIV-1. However, the para-bromine-substituted phenyl phosphate derivative of AZT, AZT-5 '- (para-bromophenylmethoxyalaniniiphosphate), 7c, was 5 times more effective than AZT in inhibiting HIV replication of TK-deficient CEM cells ( IC5o values [RT]: 0.04 μM vs 0.2 μM). none of the compounds exhibited any detectable cytotoxicity for PBMNC or CEM cells at concentrations as high as 10,000 nM, as determined through MTA. Compounds 8a-c and its compound of 3dT 3 origin were tested in a collateral comparison with d4T 1 for their ability to inhibit replication of HIV-1 in PBMNC and TK-deficient CEM T cells. The 3dT compound as well as its derivatives were less active than d4T in peripheral blood mononuclear cells as well as in TK cells deficient in TK (Table 4). Notably, in peripheral blood mononuclear cells, the ICSo values [RT] for compounds 8a-8c were greater than the IC50 value [RT] of 3dT (1.2-3.1 versus 0.7 Table 4), suggesting that these prodrugs are sufficiently stable and the independent steps of TK in its metabolism, perhaps its enzymatic hydrolysis, can be regime-limiting for the generation of active species. According to the results reported in the literature with respect to the biological activity of phenyl phosphate of d4T and AZT, phenyl phosphate derivatives of 3dT were more active than the compound of 3dT origin to inhibit replication of VI H-1 and deficient cells of TK, although it still has high micromolar values IC50 [RT] (Table 4 and Figures 4A-4F). Since compounds 8 a-8c were less active in TK-deficient CEM T cells than in PBMNC, it is postulated that the 3dT monophosphate conversion generated from these prodrugs in their active triphosphate occurs at a slower rate in the absence of TK.

Table 4 Anti-HIV activity of phenylmethoxyalaninyl phosphate derivatives of d4T, AZT and 3d T in normal peripheral blood mononuclear cells (NB PBM) and TK deficient CEM T cells.

Activity of the main compounds of d4T-5 '- (methoxyalaninyl phosphate of para-bromophenyl) and AZT-5' - (methoxyalaninylphosphate of para-bromophene lo) against HIV-2 and RTMDR-1. Principal compounds 6c and 7c were tested in collateral comparison with AZT 2 for their ability to inhibit VI H replication in RTMDR, a strain of HIV-1 and HIV-2 resistant to AZT and NN I in PBMNC cells (Table 5) . The novel derivative d4T, 6c, d4T-5 '- (para-bromophenyl methoxylaninylphosphate), had a potent antiviral activity against RTMDR-1 and moderate activity against HIV-2. However, the corresponding para-bromine substituted phenylmethoxyalaninyl phosphate derivative of AZT 7c and the AZT 2 origin were not as effective against AZT resistant or anti-H 2 RTMDR-1.

Table 5 Anti-HIV activity of major compounds 6c and 7c in HIV-2 and RTMDR -1 cells.

All data are in μM and represent concentrations required to inhibit viral replication, as measured by RT activity assays, by 50% IC5o [RT]. The computations 6a, 6b and 6c are all more potent than the d4T 1 of origin in TK cells deficient in TK, while these derivatives of d4T phenylphosphate (6a, 6b and 6c) are not more potent than the d4T 1 of origin in PBMNC infected with HIV-1 (Table 4). Comparing all derivatized phenylmethoxyalaninyl phosphate d4T, d4T-5 '- p-bromo phenylmethoxyalaninylphosphate 6c is the most potent anti-HIV agent in TK-deficient CEM cells. This observation can be attributed to the para-bromo substituent on the phenyl portion of 6c, which improves the ability of its phosphorus to undergo hydrolysis due to the electron withdrawing property of the bromo substituent (Figure 2) and results in the generation of substantially higher amounts of the key metabolite, d4T monophosphate in deficient CEM T cells from TK (Mclntee et al., 1997, J. Med. Clhem. 40: 3233: 3331). The potency of phenyl, methoxyphenyl and bromophenyl phosphate derivatives of AZT in TK-deficient CEM cells also followed the trend as that of d4T derivatives mainly 7c (bromophenyl) >7a (phenyl) > 7b (methoxyphenyl). However, among the three methoxyalaninyl phosphate derivatives of AZT (7a, 7b and 7c), only 7c showed a higher potency than AZT in TK-deficient CEM cells (IC50 values: 40 nM vs 200 nM). For phenylmethoxyalaninylphosphate derivatives of 3dT (Table 4) the presence of an electron withdrawing substituent in the para position of the phenyl portion was expected to increase the rates of hydrolysis of the substituted phenoxy group in compound 8c (eg, B a C in Figure 2). However, 8c was no more active than compound 8a without substitution for or compound 8b with a substituent for electron donor, accelerating the hypothesis that the first step of carboxysterase-dependent hydrolysis in its metabolism (for example A to B in Figure 2) plays a critical and regime-limiting role for the generation of 3dT active metabolites. It is postulated that the compounds 8a, 8b and 8c can serve as relatively poor substrates for the putative carboxyesterase responsible for their hydrolysis according to the proposed metabolic pathway for phenylmethoxyalaninylphosphate derivatives of nucleoside analogues (Figure 2). The aryl phosphate derivatives of 3dT did not behave as one might have expected from the published work regarding the metabolism and activity of the prodrug forms of a very similar d4T nucleoside analogue. Surprisingly, 3dT aryl phosphate derivatives did not produce a promising Anti-VI H activity in normal peripheral blood mononuclear cells infected with VI H-1 or TK-deficient CEM T cell line. In summary in, d4T-5 '- [p-bromo phenylmethoxyalaninyl phosphate] 6c and AZT-5' - [p-bromo phenylmethoxyalaninyl phosphate] 7c were identified as anti-VI H active agents, which potentially inhibit the replication of VI H in cells T CEM deficient in TK without any detectable cytotoxicity. In addition, the novel d4T 6c derivative had potent antiviral activity against RTMDR-1, a strain of VI H-1 resistant to AZT and NN I, and a moderate activity against VI H-2. In contrast to these 3dT derivatives, the 3dT-5 '- (para-bromo phenylmethoxyalaninyl phosphate) derivative, showed no significant anti-VI H activity in PBMNC or TK-deficient CEM T cells. For knowledge, this is the first report of comprehension of a relation of activity of structure previously not appreciated determining the presence of phenyl phosphate derivatives of d4T and AZT. Another development of the major compounds 6c and 7c may provide the basis for the design of effective strategies for the treatment of VI H capable of inhibiting HIV replication in TK-deficient cells. Although a detailed description of the present invention has been provided above, the invention is not limited thereto. The invention described herein can be modified to include alternative modalities as will be apparent to those skilled in the art. All these alternatives must be considered within the spirit and scope of the invention as claimed below.

Claims (10)

1 . A method for inhibiting VI H transcriptase activity in cells infected with VI H, which comprises administering to the infected cells an effective inhibitory amount of a compound of the formula: (0 where Y is oxygen or sulfur; Ri is monosubstituted aryl with BR, Cl, or I; R2 is a nucleoside of formula II or ll: (II) (l l l) wherein R6 is purine or pyrimidine; R7, Rβ. R9, Rio, R11 and R2 are independently hydrogen, hydroxy, halogen, azido, -NO2, -NR13R14, or -N (OR? 5) Ri6, wherein R13, R14, is and íe are independently hydrogen, acyl, alkyl, or cycloalkyl; R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R can be taken together to form a proline or hydroxyproline side chain; R5 is hydrogen, alkyl, cycloalkyl, or aryl; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein Y is oxygen.

3. The method according to claim 1, wherein Ri is bromophenyl.

4. The method according to claim 1, wherein R6 is thymine, cytosine or uracil.

5. The method according to claim 1, wherein R4 is a side chain of alanine or tryptophan.

6. A useful method for inhibiting HIV reverse transcriptase in HIV-infected cells, which comprises administering to the infected cells an inhibitory amount of a compound of formula IV or V: (IV) (V) wherein X is a group of removal of electrons.

7. The method according to claim 6, wherein X is halogen or NO2.

8. The method according to claim 7, wherein X is bromine.

9. The method according to claim 8, wherein bromine is substituted with para.

10. A method for inhibiting HIV reverse transcriptase in cells infected with VI H, which comprises administering to the infected cells an inhibitory amount of a compound of the formula: (YOU where X is an electron withdrawing group; and íe is an amino acid residue. eleven . The method according to claim 10, wherein R18 is -NHCH (CH3) COOCH3. 12. A method for inhibiting VI H replication in a host cell, which comprises contacting the host cell with an inhibitory amount of a compound of formula IV or V: (IV) (V) where X is an electron withdrawing group. 13. The method according to claim 12, wherein X is halogen or NO2. 14. The method according to claim 13, wherein X is bromine. 15. The method according to claim 14, wherein bromine is substituted with para. 16. A method for inhibiting HIV replication in host cells, which comprises administering to the cells an inhibitory amount of a compound of the formula: (VD wherein X is an electron withdrawing group, and R2 is an amino acid residue 17. The method according to claim 16, wherein R18 is -N HCH (CH3) COOCH3 18. A composition comprising an amount effective to inhibit HIV replication in a host cell of a compound of the formula: (i) where Y is oxygen or sulfur; R-t is monosubstituted aryl with BR, Cl, or I; R2 is a nucleoside of formula II or III: (I I) (I 1) wherein R 6 is purine or pyrimidine; R7, Rs, R9, R10, R11 and R? 2 are independently hydrogen, hydroxy, halogen, azido, -NO2, -N R13R1, or -N (OR? 5) Ri6, wherein R13, R1, R15 and Rie are independently hydrogen, acyl, alkyl, or cycloalkyl; R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R4 can be taken together to form a proline or hydroxyproline side chain; R5 is hydrogen, alkyl, cycloalkyl, or aryl; or a pharmaceutically acceptable salt or ester thereof; and a pharmaceutically acceptable carrier, auxiliary or diluent. 19. The composition according to claim 18, where Y is oxygen. 20. The composition according to claim 18, wherein R1 is bromophenyl. 21. The composition according to claim 18, wherein R6 is thymine, cytosine or uracil. 22. The composition according to claim 18, wherein R4 is a side chain of alanine or tryptophan. 23. A composition for inhibiting HIV replication in a host cell, comprising an amount effective to inhibit the replication of HIV in a host cell of a compound of formula IV or V: (TV) (V) wherein X ei; an electron withdrawing group and a pharmaceutically acceptable vehicle. 24. The composition according to claim 23, wherein X is halogen or NO2. 25. The composition according to claim 24, wherein X is bromine. 26. The composition according to claim 25, wherein the bromine is substituted with para. 27. A composition for inhibiting the replication of HIV in a host cell, comprising an amount effective to inhibit HIV replication in a host cell of a compound of the formula: (VINE wherein R18 is an amino acid residue and a pharmaceutically acceptable carrier. 28. The composition according to claim 27, wherein R18 is -N HCH (CH3) COOCH3. 29. A composition useful for inhibiting the replication of VI H in a host cell, comprising an amount effective to inhibit the replication of VI H in a host cell of a compound of the formula: (Vffl) wherein X is an electron withdrawing group substituted in the position for ortho and a pharmaceutically acceptable carrier. 30. The composition according to claim 29, wherein X is halogen or NO2. 31. A compound of the formula: (l) where Y is oxygen or sulfur; Ri is aryl monosubstituted with Br, Cl, or I; R2 is a nucleoside of formula II or III: (I I) (M I) wherein R6 is purine or pyrimidine; R7, R8, R9, Rio, R1 1 and R? 2 are independently hydrogen, hydroxy, halogen, azido, -NO2, -NR? 3R? 4, or -N (OR? 5) Ri6, wherein R13, R14, R 5 and R 16 are independently hydrogen, acyl, alkyl, or cycloalkyl; R3 is hydrogen acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R4 can be taken together to form a proline or hydroxyproline side chain; R5 is hydrogen, alkyl, cycloalkyl, or aryl; or a pharmaceutically acceptable salt or ester thereof. 32. The compound according to claim 31, having the formula: where X is chosen from Br and Cl; or a pharmaceutically acceptable salt or ester thereof. 33. A compound of the formula: where Y is oxygen or sulfur; Ri is monosubstituted aryl with BR, Cl, or I, NO2 or OMe; R6 is purine or pyrimidine; R11 and R12 are independently hydrogen, hydroxy, halogen, azido, -NO2, -NR13R14, or -N (OR? 5) Ri6, wherein R13, R14, R15 and R16 are independently hydrogen, acyl, alkyl, or cycloalkyl; R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino acid; or R3 and R4 can be taken together to form a proline or hydroxyproline side chain; and R5 is hydrogen, alkyl, cycloalkyl, or aryl; or a pharmaceutically acceptable salt or ester thereof. 34. The compound according to claim 33, having the formula: or a pharmaceutically acceptable salt or ester thereof. 35. The compound according to claim 34, having the formula: or a pharmaceutically acceptable salt or ester thereof. 36. A method for inhibiting HIV reverse transcriptase activity in HIV-infected cells, comprising administering to the infected cells an effective inhibitory amount compound of any of claims 31 to 35. A composition comprising an effective amount for inhibit HIV replication in a host cell of the compound of any of claims 31 to 35.