Antiviral Chemistry & Chemotherapy 13:

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1 Antiviral Chemistry & Chemotherapy 13: Aryl-substituted and benzo-annulated cyclosal-derivatives of 2,3 -dideoxy-2,3 -didehydrothymidine monophosphate correlation of structure, hydrolysis properties and anti-hiv activity Christian Ducho 1, Jan Balzarini 2, Lieve aesens 2, Erik De Clercq 2 and Chris Meier 1 * 1 Institut für rganische Chemie, Universität Hamburg, Hamburg, Germany 2 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium *Corresponding author. Tel: ; Fax: ; chris.meier@chemie.uni-hamburg.de The synthesis of phenyl-substituted and benzoannulated cyclosal phosphate triesters of the nucleoside analogue 2,3 -dideoxy-2,3 -didehydrothymidine (d4t, Zerit ) as lipophilic, membrane-soluble pronucleotides is described. The cyclosal moiety was introduced by using cyclic chlorophosphite agents prepared from phenylsubstituted saligenin derivatives and orthohydroxymethylated naphthols, respectively. Hydrolysis studies (HLC analysis) of the triesters 2, 3 showed a range of hydrolytic stability from 1.4 h up to 5.1 h and the stability could be correlated with the substitution pattern in the cyclosal moiety. A slight decrease of their stability was observed, if phenyl-substituted derivatives were hydrolyzed in human CEM/ cell extracts. D4T and thymine, possible products of enzymatic cleavage of the pronucleotides, were not detected in the cell extracts. A further investigation of the hydrolysis process was performed by 31 -MR spectroscopy. This technique allowed a precise monitoring of the degradation products and the exact determination of the product ratio. Finally, the newly synthesized compounds were tested concerning their antiviral activity against HIV in vitro. A strong correlation of the hydrolysis properties and the antiviral activity was found. 3-phenyl-cycloSal-d4TM showed a threefold increase in its anti-hiv-1 activity and retained full activity in thymidine kinase (TK) deficient cells, indicative of a successful TK-bypass. Keywords: d4t, HIV, TK bypass, pronucleotides, cyclosal nucleotides, 31 -MR Introduction The nucleoside analogue 2,3 -dideoxy-2,3 -didehydrothymidine (d4t 1, Zerit ) is well known for its anti- HIV activity, serving as an inhibitor of the viral enzyme reverse transcriptase (Riddler et al., 1995). evertheless, the metabolism of d4t to the biologically active d4t triphosphate (d4tt) is critical. articularly the first metabolic step, the phosphorylation of 1 to d4t monophosphate (d4tm) by the cellular thymidine kinase (TK), is rather poor and inefficient (Balzarini et al., 1989; Balzarini, 1994). However, a direct administration of d4tmonophosphate (M) to the target cells is impossible because of its poor membrane permeability and high lability towards unspecific dephosphorylation. Therefore, different approaches have been reported describing the design of lipophilic d4tm derivatives as pronucleotides for a selective intracellular d4tm delivery (TK-bypass; Meier et al., 1997a; Wagner, 2000). To qualify as a suitable nucleotide prodrug, the masked nucleotide has to be lipophilic, to allow passive diffusion through the cell membrane and the blood-brain barrier and should be able to deliver the nucleotide intracellularly upon release of non-toxic masking groups hydrolytically or enzymatically. Meier et al. (1996, 2002) have developed the cyclosaligenyl (cyclosal) concept, which is based on a purely hydrolytic cleavage of the pronucleotides under physiological ph conditions. The cyclosal nucleotide prodrugs differ from the other prodrug approaches in that they may successfully deliver nucleotides by chemical activation, involving a highly selective coupled two-step cascade mechanism. As a result, the cyclosal-concept requires only one activation step to deliver the nucleotide, and only one masking unit per nucleotide is required. In earlier work, we demonstrated that the half-life of the nucleotide release can be fine-tuned by the nature of the substituent on the cyclosal part of the molecule. The successful intracellular release of nucleotides from the cyclosal nucleotide prodrugs has been demonstrated for the cyclosal-d4tm prodrug (Meier et al., 1997b, 1998a; Balzarini et al., 2000). Also, markedly improved 2002 International Medical ress /02/$

2 C Ducho et al. Figure 1. Structural formulae of target triesters 2 and 3 and reference compounds 9 5 X 3 2a X=3-C 6 H 5 ; 3-phenyl-cycloSal-d4TM 2b X=5-C 6 H 5 ; 5-phenyl-cycloSal-d4TM 9a X=H; cyclosal-d4tm 9b X=3-CH 3 ; 3-methyl-cycloSal-d4TM 9b X=5-CH 3 ; 5-methyl-cycloSal-d4TM benzo[b]-cyclosal-d4tm 3b benzo[a]-cyclosal-d4tm 3a benzo[c]-cyclosal-d4tm 3c antiviral properties of cyclosal nucleotide prodrugs have been found for a variety of nucleosides, including 2,3 - dideoxyadenosine (dda), 2,3 -dideoxy-2,3 -didehydroadenosine (d4a), 2 -fluoro-dda (Meier et al., 1997c, 1999a,b), acyclovir (Meier et al., 1998b) (E)-5-(2-bromovinyl)-2 -deoxyuridine (BVDU) (Meier et al., 2001a, b) and carbovir (CBV)/abacavir (Balzarini et al., 2002). The successful d4tm delivery has been shown in studies with radiolabelled cyclosal-d4tm compounds (Balzarini et al., 2000). However, the cyclosal approach could not be successfully applied to 3 -azidothymidine (AZT) (Meier et al., 1998c; Balzarini et al., 1999). In this study we describe the synthesis and characterization of phenyl-substituted and benzo-annulated (naphthyl) cyclosal-d4tm phosphate triesters. The target molecules have been the 3- and 5-phenyl-cycloSal-d4TMs 2a, b as well as the benzo[a]-, benzo[b]- and benzo[c]-annulated cyclosal-d4tms 3a c (Figure 1). It should be pointed out that the nomenclature of the benzo-annulated derivatives is not in accordance to the International Union of ure and Applied Chemistry (IUAC) nomenclature for reasons of simplification. Materials and methods: chemistry MR spectra were recorded using Bruker AC 250-, Bruker AMX 400 and Bruker DRX 500 Fourier transform spectrometers. All 1 H and 13 C MR chemical shifts (δ) are quoted in parts per million (p.p.m.) and calibrated on solvent signals. The 31 MR chemical shifts are quoted in p.p.m. using H 3 4 as the external reference. Coupling constants ( J) are given in Hz. The spectra were recorded at room temperature. Electron impact mass spectra were measured on a VG Analytical VG/70-250S spectrometer (double focussing). FAB high resolution (HR) mass spectra were recorded on a VG Analytical S spectrometer using an MCA method and polyethylene glycol as support. Merck precoated 60 F 254 plates with a 0.2 mm layer of silica gel were used for thin layer chromatography (TLC). All preparative TLCs were performed on a chromatotron (Harrison Research, Model 7924T) using glass plates coated with 1 mm or 2 mm layers of Merck 60 F 254 silica gel containing a fluorescent indicator. Analytical high performance liquid chromatography (HLC) was done on a Merck-Hitachi HLC system (D-7000) equipped with a LiChroCART column containing reversed phase silica gel Lichrospher 100 R 18 (5 µm). reparative HLC was carried out on a Merck-Hitachi system with an L-6250 Intelligent ump (equipped with a 5 ml injection device), a LaChrom L-7400 UV detector and a D-2500A integrator. The column was a Merck Hibar RT containing reversed phase silica gel Lichrospher 100 R 18 (5 µm) (Merck, Darmstadt, Germany). Separations were performed with a flow of 12 ml/min and detection at 260 nm wavelength. The lyophilized products 2a, b and 3a c did not give useful microanalytical data most probably due to incomplete combustion of the compounds but were found to be pure by rigorous HLC analysis (gradient of 5 100% acetonitrile in water within 25 min, flow 0.5 ml/min). All reactions were carried out under an atmosphere of dry nitrogen except for the synthesis of 4a, b, 5a and 8. Solvents used were commercially available dry solvents stored under argon and over molecular sieve (Fluka). Diethyl ether was dried over sodium/benzophenone and distilled under nitrogen. Diastereomeric ratios of the cyclosal phosphate triesters were determined by integration of the 31 MR spectra if two signals were observed. General procedure for the preparation of cyclosal d4t monophosphates (cyclosald4tms) (2a, b and 3a c) The chemical synthesis of cyclosal-d4t monophosphates has been published before (Meier et al., 1998). Acetonitrile or a dimethylformamide/tetrahydrofuran (DMF/THF) mixture (2:1 v/v) were used as solvents. The chlorophosphite/t-butylhydroperoxide (TBH) method has been applied at 20 C. roducts 2 and 3 were isolated as colourless foams after chromatographic purification and lyophilization. 3-phenyl-cycloSal d4t monophosphate (3- phenyl-cyclosal-d4tm) (2a) Quantities: d4t 1 (1.0 equiv., 100 mg, 0.45 mmol), acetonitrile (10 ml), DIEA (2.0 equiv., 0.16 ml, 0.91 mmol), International Medical ress

3 ovel cyclosal prodrugs of d4t crude product 6a (236 mg, dissolved in 1.9 ml acetonitrile), TBH (5 6 M solution in n-decane, 0.30 ml, 1.5 mmol). After chromatography, the product 2a was isolated as a mixture of two diastereomers in a ratio of 1.0:0.7 (117 mg, 56%); R f : 0.53 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ (s, 1 1H, 1 H), (s, 1 1H, 1 H), (m, 2 8H, 2 8 aryl-h), 7.17 (q, 1 1H, 1 thymine-h6, J=1.3), 7.17 (s, 1 1H, 1 thymine-h6), (m, 2 1H, 2 H1 ), 6.33 (dd, 2 1H, 2 H3, J=6.1, J=1.7), 6.01 (dd, 1 1H, 1 H2, J=5.9, J=1.7), 5.94 (dd, 1 1H, 1 H2, J=5.6, J=1.7), 5.58 (dd, 1 1H, 1 benzyl-h, J=14.3, J =2.8), 5.54 (dd, 1 1H, 1 benzyl-h, J=14.3, J =1.8), 5.49 (d, 1 1H, 1 benzyl-h, J=13.5), 5.45 (d, 1 1H, 1 benzyl-h, J=13.5), (m, 2 1H, 2 H4 ), 4.30 (ddd, 2 2H, 2 H5, J=12.5, J=7.0, J =3.1), 1.56 (s, 1 3H, 1 thymine-ch 3 ), 1.53 (s, 1 3H, 1 thymine-ch 3 ); 13 C MR (100 MHz, DMS-d 6 ): δ (2 thymine-c4), (2 thymine-c2), (1 thymine-c6), (1 thymine-c6), ( J=6.6, 2 aryl-c2), (1 C3 ), (1 C3 ), (2 aryl-c4), (2 aryl-c1 ), (2 aryl-c3, 2 aryl-c5 ), (1 aryl-c2, 1 aryl-c6 ), (1 aryl-c2, 1 aryl-c6 ), (2 aryl-c6), (1 C2 ), (1 C2 ), (2 aryl-c4 ), (1 aryl-c5), (1 aryl-c5), (2 aryl-c1), (2 aryl-c3), (2 thymine-c5), (1 C1 ), (1 C1 ), (1 C4 ), (1 C4 ), (2 C5 ), (2 benzyl-c), (1 thymine- CH 3 ), (1 thymine-ch 3 ); 31 MR (202 MHz, DMS-d 6 ): δ 7.17, 7.08; MS (FAB-HR): m/z found , calc [M+H] + ; HLC: t R =14.0 min. 5-phenyl-cycloSal d4t monophosphate (5-phenyl-cycloSal-d4TM) (2b) Quantities: d4t 1 (1.0 equiv., 100 mg, 0.45 mmol), acetonitrile (10 ml), DIEA (2.0 equiv., 0.16 ml, 0.91 mmol), crude product 6b (236 mg, dissolved in 2.1 ml acetonitrile), TBH (5 6 M solution in n-decane, 0.30 ml, 1.5 mmol). After chromatography, the product 2b was isolated as a mixture of two diastereomers (105 mg, 50%); R f : 0.56 (CH 2 /methanol 9:1 v/v); 1 H MR (500 MHz, DMS-d 6 ): δ (s, 2 1H, 2 H), (m, 2 4H, 2 aryl-h4, 2 aryl-h6, 2 aryl-h2, 2 aryl-h6 ), 7.49 (dd, 2 2H, 2 aryl-h3, 2 aryl-h5, J=7.6, J=7.6), 7.40 (t, 2 1H, 2 aryl-h4, J=7.6), 7.25 (d, 1 1H, 1 aryl- H3, J=11.4), 7.24 (q, 1 1H, 1 thymine-h6, J=1.1), 7.21 (q, 1 1H, 1 thymine-h6, J=1.1), 7.21 (d, 1 1H, 1 aryl- H3, J=8.5), (m, 1 1H, 1 H1 ), (m, 1 1H, 1 H1 ), 6.46 (ddd, 1 1H, 1 H3, J=6.0, J=1.6, J=1.6), 6.41 (ddd, 1 1H, 1 H3, J=6.0, J=1.6, J=1.6), 6.05 (m, 2 1H, 2 H2 ), 5.60 (dd, 1 1H, 1 benzyl-h, J=17.3, J =6.6), 5.57 (dd, 1 1H, 1 benzyl-h, J=17.3, J =6.6), 5.50 (d, 1 1H, 1 benzyl-h, J=10.4), 5.47 (d, 1 1H, 1 benzyl- H, J=10.4), (m, 2 1H, 2 H4 ), (m, 2 2H, 2 H5 ), 1.71 (d, 1 3H, 1 thymine-ch 3, J=1.1), 1.64 (d, 1 3H, 1 thymine-ch 3, J=1.1); 13 C MR (100 MHz, DMS-d 6 ): δ (2 thymine-c4), (2 thymine-c2), (2 aryl-c2), (2 aryl- C1 ), (2 thymine-c6), (1 aryl-c5), (1 aryl-c5), (1 C3 ), (1 C3 ), (2 aryl-c3, 2 aryl-c5 ), (1 aryl-c4), (1 aryl-c4), (2 aryl-c4 ), (1 C2 ), (1 C2 ), (2 aryl-c2,2 aryl-c6 ), (2 aryl- C6), (2 aryl-c1), (1 aryl-c3), (1 aryl-c3), (2 thymine-c5), (2 C1 ), ( J=2.5, 1 C4 ), ( J=1.5, 1 C4 ), (m, 2 C5, 2 benzyl-c), (1 thymine-ch 3 ), (1 thymine-ch 3 ); 31 MR (202 MHz, DMS-d 6 ): δ 8.16; MS (FAB-HR): m/z found , calc [M+H] + ; HLC: t R =14.9 min. Benzo[a]-cycloSal d4t monophosphate (benzo[a]-cyclosal-d4tm) (3a) Quantities: d4t 1 (1.0 equiv., 207 mg, 0.92 mmol), DMF/THF 2:1 v/v (6 ml), DIEA (2.1 equiv., 0.33 ml, 1.90 mmol), crude product 7a (440 mg, dissolved in 2.6 ml DMF/THF 2:1 v/v), TBH (3.4 equiv., 5.5 M solution in n-decane, 0.56 ml, 3.1 mmol). The purification by preparative TLC turned out to be difficult, so that parts of the product had to be purified by preparative HLC (eluent acetonitrile/water 7:10 v/v, water acidified with 0.5% glacial acetic acid). The product 3a was finally isolated as a mixture of two diastereomers in a ratio of 0.8:1.0 (101 mg, 25%); R f : 0.48 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ (s, 2 1H, 2 H), (m, 2 2H, 2 naphthyl-h1, 2 naphthyl-h4 ), 7.79 (d, 2 1H, 2 naphthyl-h5, J=8.5), (m, 2 2H, 2 naphthyl-h2, 2 naphthyl-h3 ), 7.37 (d, 1 1H, 1 naphthyl-h6, J=8.5), 7.36 (d, 1 1H, 1 naphthyl-h6, J=8.5), 7.21 (q, 1 1H, 1 thymine-h6, J=1.3), 7.19 (q, 1 1H, 1 thymine-h6, J=1.1), (m, 2 1H, 2 H1 ), 6.44 (ddd, 1 1H, 1 H3, J=6.1, J=2.3, J=1.8), 6.36 (ddd, 1 1H, 1 H3, J=5.9, J=2.3, J=1.8), 6.04 (ddd, 1 1H, 1 H2, J=5.9, J=2.3, J=1.3), 6.00 (ddd, 1 1H, 1 H2, J=6.1, J=2.3, J=1.5), (m, 2 2H, 2 benzyl-h), (m, 2 1H, 2 H4 ), (m, 2 2H, 2 H5 ), 1.62 (d, 1 3H, 1 thymine-ch 3, J=1.3), 1.54 (d, H, 1 thymine-ch 3, J=1.1); C MR (100 MHz, DMS-d 6 ): δ (2 thymine-c4), (2 thymine-c2), ( J=4.1, 2 naphthyl-c2), (2 thymine-c6), (2 naphthyl-c4), (2 C3 ), (2 naphthyl-c3), (2 naphthyl- C2 ), (2 naphthyl-c4 ), (2 C2 ), (2 naphthyl-c3 ), (2 naphthyl-c5), (2 naphthyl-c6), (2 naphthyl-c1), (2 naphthyl-c1 ), (1 thymine-c5), Antiviral Chemistry & Chemotherapy 13:1 3

4 C Ducho et al. (1 thymine-c5), (2 C1 ), (1 C4 ), (1 C4 ), (m, 2 C5, 2 benzyl-c), (1 thymine-ch 3 ), (1 thymine-ch 3 ); 31 MR (202 MHz, DMS-d 6 ): δ 8.48, 8.46; MS (FAB-HR): m/z found , calc [M+H] + ; HLC: t R =13.8 min. Benzo[b]-cycloSal d4t monophosphate (benzo[b]-cyclosal-d4tm) (3b) Quantities: d4t 1 (1.0 equiv., 102 mg, 0.46 mmol), acetonitrile (10 ml), DIEA (2.0 equiv., 0.16 ml, 0.93 mmol), crude product 7b (217 mg, dissolved in 2.3 ml acetonitrile), TBH (5 6 M solution in n-decane, 0.31 ml, 1.6 mmol). To complete the conversion of 1, 109 mg of 7b, dissolved in 1.2 ml acetonitrile, had to be added additionally. After chromatography, the product 3b was isolated as a mixture of two diastereomers in a ratio of 1.0:0.9 (66 mg, 33%); R f : 0.47 (CH 2 /methanol 9:1 v/v); 1 H MR (500 MHz, DMS-d 6 ): δ (s, 1 1H, 1 H), (s, 1 1H, 1 H), (m, 2 3H, 2 naphthyl-h6, 2 naphthyl-h1, 2 naphthyl-h4 ), 7.73 (s, 1 1H, 1 naphthyl- H3), 7.70 (s, 1 1H, 1 naphthyl-h3), 7.58 (ddd, 2 1H, 2 naphthyl-h2, J=6.9, J=6.9, J=1.3), 7.54 (ddd, 2 1H, 2 naphthyl-h3, J=6.9, J=6.9, J=1.3), 7.23 (q, 1 1H, 1 thymine-h6, J=1.3), 7.20 (q, 1 1H, 1 thymine-h6, J=1.3), (m, 2 1H, 2 H1 ), (m, 1 1H, 1 H3 ), (m, 1 1H, 1 H3 ), (m, 2 1H, 2 H2 ), 5.69 (dd, 1 1H, 1 benzyl-h, J=15.1, J =5.7), 5.66 (dd, 1 1H, 1 benzyl-h, J=15.1, J =6.3), 5.59 (dd, 1 1H, 1 benzyl-h, J=13.2, J =6.3), 5.56 (dd, 1 1H, 1 benzyl-h, J=13.2, J =6.9), (m, 2 1H, 2 H4 ), (m, 2 2H, 2 H5 ), 1.66 (d, 1 3H, 1 thymine-ch 3, J=1.3), 1.58 (d, 1 3H, 1 thymine-ch 3, J=1.3); 13 C MR (125 MHz, DMS-d 6 ): δ (1 thymine-c4), (2 thymine-c2), ( J=2.4, 2 naphthyl-c2), (2 naphthyl-c4), (2 thymine-c6), (2 C3 ), (2 naphthyl- C5), (2 naphthyl-c6), (2 C2 ), (2 naphthyl-c4 ), (2 naphthyl-c1), (2 naphthyl-c1 ), (2 naphthyl-c2 ), (2 naphthyl-c3 ), (1 naphthyl-c3), (1 naphthyl-c3), (1 thymine-c5), (1 thymine-c5), (2 C1 ), (1 C4 ), (1 C4 ), ( J=6.1, 1 C5 ), ( J=7.3, 1 C5 ), ( J=6.1, 1 benzyl-c), ( J=6.1, 1 benzyl-c), (1 thymine-ch 3 ), (1 thymine-ch 3 ); MR (202 MHz, DMS-d 6 ): δ 7.25, 7.16; MS (FAB-HR): m/z found , calc [M+H] + ; HLC: t R =13.0 min. Benzo[c]-cycloSal d4t monophosphate (benzo[c]-cyclosal-d4tm) (3c) Quantities: d4t 1 (1.0 equiv., 122 mg, 0.54 mmol), acetonitrile (12 ml), DIEA (2.0 equiv., 0.19 ml, 1.11 mmol), crude product 7c (260 mg, dissolved in 1.8 ml DMF/THF 2:1 v/v), TBH (3.3 equiv., 5.5 M solution in n-decane, 0.33 ml, 1.8 mmol). After chromatography, the product 3c was isolated as a mixture of two diastereomers in a ratio of 1.0:0.6 (143 mg, 59%); R f : 0.53 (CH 2 /methanol 9:1 v/v); 1 H MR (500 MHz, DMS-d 6 ): δ (s, 1 1H, 1 H), (s, 1 1H, 1 H), 8.01 (d, 2 1H, 2 naphthyl-h4, J=8.2), 7.99 (d, 2 1H, 2 naphthyl-h4, J=8.8), 7.82 (d, 1 1H, 1 naphthyl-h1, J=8.2), 7.77 (d, 1 1H, 1 naphthyl-h1 ), 7.64 (dd, 2 1H, 2 naphthyl-h3, J=8.2, J=8.2), 7.57 (dd, 2 1H, 2 naphthyl-h2, J=8.2, J=8.2), 7.32 (d, 1 1H, 1 naphthyl-h3, J=8.8), 7.29 (d, 1 1H, 1 naphthyl-h3, J=9.5), 7.24 (q, 1 1H, 1 thymine-h6, J=1.3), 7.20 (q, 1 1H, 1 thymine-h6, J=1.3), (m, 1 1H, 1 H1 ), (m, 1 1H, 1 H1 ), (m, 1 1H, 1 H3 ), (m, 1 1H, 1 H3 ), (m, 1 2H, 2 1H, 1 benzyl-h, 2 H2 ), 5.89 (dd, 1 1H, 1 benzyl-h, J=14.5, J =8.2), 5.86 (dd, 1 1H, 1 benzyl-h, J=14.5, J =8.2), (m, 2 1H, 2 H4 ), (m, 2 2H, 2 H5 ), 1.70 (s, 1 3H, 1 thymine-ch 3 ), 1.63 (s, 1 3H, 1 thymine-ch 3 ); 13 C MR (125 MHz, DMS-d 6 ): δ (1 thymine-c4), (1 thymine-c4), (1 thymine-c2), (1 thymine-c2), ( J=6.1, 1 naphthyl-c2), ( J=6.1, 1 naphthyl-c2), (1 thymine-c6), (1 thymine-c6), (1 C3 ), (1 C3 ), (1 naphthyl-c4), (1 naphthyl-c4), (2 naphthyl-c4 ), (2 naphthyl-c6), (1 naphthyl-c3 ), (1 naphthyl-c3 ), (1 C2 ), (1 C2 ), (2 naphthyl-c5), (2 naphthyl-c2 ), (1 naphthyl-c1 ), (1 naphthyl-c1 ), (1 naphthyl-c3), (1 naphthyl-c3), (1 naphthyl-c1), (1 naphthyl-c1), (1 thymine-c5), (1 thymine-c5), (1 C1 ), (1 C1 ), (1 C4 ), (1 C4 ), ( J=6.1, 1 C5 ), ( J=6.1, 1 C5 ), ( J=7.3, 1 benzyl-c), ( J=8.5, 1 benzyl-c), (1 thymine-ch 3 ), (1 thymine- CH 3 ); 31 MR (202 MHz, DMS-d 6 ): δ 9.50, 9.48; MS (FAB-HR): m/z found , calc [M+H] + ; HLC: t R =12.9 min, 13.1 min. 3-phenyl salicyl alcohol (4a) In a Dean-Stark apparatus, 2-hydroxybiphenyl (1.0 equiv., 2.98 g, 17.3 mmol) was dissolved in 100 ml toluene and phenylboronic acid (1.2 equiv., 2.56 g, 21.0 mmol), propionic acid (0.5 equiv., 653 mg, 8.81 mmol) and paraformaldehyde (2.0 equiv., 1.02 g, 33.9 mmol) were added. The solution was heated under reflux for 8 h, while more paraformaldehyde (8.2 equiv., 4.23 g, 141 mmol) was added in four portions. The reaction was monitored by TLC (CH 2 /methanol 9:1 v/v). After complete conver International Medical ress

5 ovel cyclosal prodrugs of d4t sion of the 2-hydroxybiphenyl, the reaction mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was dissolved in CH 2 and washed with water twice. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by preparative TLC (CH 2 as eluent). After evaporation of the solvent, the resulting pure product was dissolved in 12 ml THF. After cooling to 0 C, 12 ml of a 30% solution of hydrogen peroxide in water were added and the solution was stirred for 35 min at 0 C. After dilution with 50 ml water and reduction of the excess peroxide with sodium hydrogen sulfite (39% solution in water), the aqueous phase was extracted with diethyl ether. The combined organic solutions were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was recrystallized from n-pentane to yield a colourless solid (2.12 g, 61%); R f : 0.83 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ (br, 1H, phenol-h), 7.52 (dd, 2H, H2, H6, J=7.1, J=1.3), 7.43 (dd, 2H, H3, H5, J=7.1, J=7.1), 7.33 (tt, 1H, H4, J=7.1, J=1.3), 7.24 (dd, 1H, H4, J=7.5, J=1.5), 7.15 (dd, 1H, H6, J=7.5, J=1.5), 6.93 (dd, 1H, H5, J=7.5, J=7.5), (br, 1H, benzyl-h), 4.67 (s, 2H, benzyl-h); 13 C MR: (100 MHz, DMS-d 6 ): δ (C2), (C1 ), (C2,C6 ), (C1), (C3), (C6), (C3,C5 ), (C4), (C4 ), (C5), (benzyl-c); MS (EI + ): m/z [M]. 5-phenyl salicyl alcohol (4b) 4-hydroxybiphenyl (2.86 g, 16.8 mmol) and potassium hydroxide (15.2 g, 271 mmol) were dissolved in 180 ml of a 37% aqueous formaldehyde solution at 0 C. Afterwards, the reaction mixture was slowly warmed to room temperature (2.3 h) and stirred for 24 h. The reaction was monitored by TLC (CH 2 /methanol 9:1 v/v) and finally, after conversion of about one third of the educt, stopped by addition of 10% aqueous hydrochloric acid up to ph 3. CH 2 was added, and the aqueous phase extracted with CH 2. The combined organic solutions were dried over sodium sulfate and evaporated under reduced pressure. The resulting crude product was purified by column chromatography (CH 2 with a methanol gradient from 0 to 1%) to yield a colourless solid (1.25 g, 37%); R f : 0.59 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ (br, phenol-h), 7.61 (d, H6, J=2.3), 7.58 (dd, 2H, H2, H6, J=7.8, J=1.3), 7.43 (dd, 2H, H3, H5, J=7.8, J=7.8), 7.37 (dd, 1H, H4, J=8.2, J=2.3), 7.29 (t, 1H, H4, J=7.4), 6.87 (d, 1H, H3, J=8.2), (br, 1H, benzyl-h), 4.56 (s, 2H, benzyl-h); 13 C MR (100 MHz, DMS-d 6 ): δ (C2), (C1 ), (C5), (C1), (C3,C5 ), (C4 ), (C2,C6 ), (C6), (C4), (C3), (benzyl-c); MS (EI + ): m/z [M]. Methyl-1-hydroxy-2-naphthoate (8) 1-hydroxy-2-naphthoic acid (1.0 equiv., 18.9 g, 100 mmol) was dissolved in 250 ml methanol. Concentrated sulfuric acid (50 ml) was added slowly, and the resulting solution was heated under reflux for 20 h. After cooling to room temperature, the reaction mixture was poured into 1 l ice/water. The resulting colourless precipitate was filtered off and washed with cold water. Recrystallization from methanol/water afforded 8 as a slightly grey solid (17.2 g, 85%); R f : 0.81 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ (s, 1H, phenol-h), 8.32 (d, 1H, H8, J=7.9), 7.93 (d, 1H, H5, J=8.1), 7.76 (d, 1H, H3, J=8.7), 7.71 (ddd, 1H, H6, J=8.1, J=8.1, J=1.1), 7.62 (ddd, 1H, H7, J=7.9, J=7.9, J=1.0), 7.44 (d, 1H, H4, J=8.7), 3.98 (s, 3H, CH 3 ); 13 C MR (125 MHz, DMS-d 6 ): δ (C=), (C1), (C4a), (C6), (C5), (C7), (C3), (C8a), (C8), (C4), (C2), (CH 3 ); MS (EI + ): m/z [M]. 2-(hydroxymethyl)-1-naphthol (5a) Lithium aluminium hydride (560 mg, 14.8 mmol) was suspended in 50 ml dry THF and a solution of 8 (2.0 g, 9.89 mmol) in 30 ml dry THF was added dropwise over a 30 min period under vigorous stirring at room temperature. After completion of the addition, the mixture was stirred for 2 h at room temperature and for 1 h under reflux. After cooling to 0 C, 120 ml diethyl ether were added for dilution and excess lithium aluminium hydride was destroyed by addition of 2 hydrochloric acid until ph 5. Water was added, and the aqueous phase was extracted with ether. The combined organic solutions were dried over sodium sulfate and evaporated under reduced pressure to afford a yellow brown oil (1.79 g, 100% yield: 1.72 g) which was primarily contaminated with salts and degradation products of 5a. This oil was directly used for the synthesis of the phosphitylating agent 7a without further purification due to the high lability of the diol 5a;Rf: 0.63 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ 9.33 (s, 1H, phenol-h), (m, 1H, H8), (m, 1H, H5), (m, 4H, H3, H4, H6, H7), 5.44 (t, 1H, benzyl- H, J=5.0), 4.79 (d, 2H, benzyl-h, J=5.0); 13 C MR (125 MHz, DMS-d 6 ): δ (C1), (C4a), 127,58 (C5), (C8a), (C3), (C7), (C6), (C8) (C2), (C4), (benzyl-c). 3-(hydroxymethyl)-2-naphthol (5b) Lithium aluminium hydride (710 mg, 18.7 mmol) was suspended in 50 ml dry THF and a solution of methyl 3-hydroxy-2-naphthoate (1.88 g, 9.31 mmol) in 30 ml dry Antiviral Chemistry & Chemotherapy 13:1 5

6 C Ducho et al. THF was added dropwise over a 30 min period under vigorous stirring at room temperature. After completion of the addition, the mixture was stirred for 3 h at room temperature. After cooling to 0 C, the reaction mixture was diluted by addition of 80 ml diethyl ether and excess lithium aluminiumhydride was destroyed by adding 10% hydrochloric acid until ph 4. Water was added, and the aqueous phase was extracted with ether vigorously. The combined organic solutions were dried over sodium sulfate and evaporated under reduced pressure to afford a slightly brown solid (1.60 g, 99%); R f : 0.58 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ 9.81 (s, 1H, phenol-h), 7.83 (s, 1H, H4), 7.78 (d, 1H, H5, J=8.1), 7.66 (d, 1H, H8, J=7.9), 7.35 (ddd, 1H, H7, J=7.9, J=7.9, J=1.0), 7.26 (ddd, 1H, H6, J=8.1, J=8.1, J=1.3), 7.12 (s, 1H, H1), 5.17 (s, 1H, benzyl-h), 4.66 (s, 2H, benzyl-h); 13 C MR (100 MHz, DMS-d 6 ): δ (C2), (C8a), (C4a), (C3), (C5), (C8), (C7), (C4), (C6), (C1), (benzyl-c); MS (EI + ): m/z [M]. 1-(hydroxymethyl)-2-naphthol (5c) 2-naphthol (5.0 g, 34.7 mmol) was dissolved in exactly 36.7 ml of a 1 aqueous sodium hydroxide solution at 0 C. To this solution, 3.72 ml of a 37% aqueous formaldehyde solution were added within 5 min at 0 C. After further stirring for 1 h at 0 C, 1 hydrochloric acid was added for neutralization. The resulting precipitate was filtered off, washed with water and dried under reduced pressure to yield 5c as a slightly brown solid (5.73 g, 95%); R f : 0.49 (CH 2 /methanol 9:1 v/v); 1 H MR (400 MHz, DMS-d 6 ): δ 8.06 (d, 1H, H8, J=8.6), 7.78 (d, 1H, H5, J=7.9), 7.71 (d, 1H, H4, J=8.9), 7.46 (ddd, 1H, H7, J=8.6, J=8.6, J=1.3), 7.29 (ddd, 1H, H6, J=7.9, J=7.9, J=0.8), 7.16 (d, 1H, H3, J=8.9), 4.95 (s, 2H, benzyl-h); 13 C MR (125 MHz, DMS-d 6 ): δ (C2), (C8a), (C4), (C4a), (C5), (C7), (C8), (C6), (C3), (C1), (benzyl-c); MS (EI + ): m/z [M]. General procedure for the preparation of the saligenyl chlorophosphites (6a, b and 7a c) A solution or suspension of 1.0 equiv. of the salicyl alcohol derivative or hydroxymethylated naphthol in dry diethyl ether was cooled to 20 C. After addition of 1.2 equiv. of freshly distilled phosphorus(iii) chloride and stirring for 5 20 min at 20 C, a solution of 2.3 equiv. dry pyridine in dry ether was added within h at 20 C. After completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for 1 2 h to be furthermore kept at 20 C overnight for a complete precipitation of pyridinium chloride. Filtration under a nitrogen atmosphere and evaporation of the filtrate under reduced pressure afforded 6a, b and 7a c as crude products to be directly used for the synthesis of the cyclosal phosphate triesters without further purification. 3-phenyl saligenyl chlorophosphite (6a) Quantities: 4a (1.0 g, 4.99 mmol), dissolved in 30 ml ether, phosphorus(iii) chloride (0.51 ml, 5.77 mmol), pyridine (0.93 ml, 11.6 mmol), dissolved in 3.9 ml ether. Reaction periods: 15 min after addition of phosphorus(iii) chloride, 3 h for the addition of the pyridine solution, 1.3 h stirring at room temperature. This afforded racemic 6a as a crude yellow oil (1.26 g, 100% yield: 1.32 g); 1 H MR (400 MHz, CDCl 3 ): δ (m, 6H, H4, H2, H3, H4, H5,H6 ), 7.08 (dd, 1H, H5, J=7.6, J=7.6), 6.89 (ddd, 1H, H6, J=7.6, J=0.8, J=0.8), 5.42 (d, 1H, benzyl-h, J=14.1), 5.03 (d, 1H, benzyl-h, J=14.1); 13 C MR (100 MHz, CDCl 3 ): δ (C2), (C1 ), (C4), (C3, C5 ), (C2, C6 ), (C6), (C4 ), (C5), (C1), (C3), ( J=2.0, benzyl-c); 31 MR (101 MHz, CDCl 3 ): δ phenyl saligenyl chlorophosphite (6b) Quantities: 4b (800 mg, 4.0 mmol), suspended in 60 ml ether, phosphorus(iii) chloride (0.41 ml, 4.63 mmol), pyridine (0.75 ml, 9.27 mmol), dissolved in 3.2 ml ether. Reaction periods: 20 min. after addition of phosphorus(iii) chloride, 3 h for the addition of the pyridine solution, 2 h stirring at room temperature. This afforded racemic 6b as a crude yellow brown oil (1.13 g, 100% yield: 1.06 g); 1 H MR (400 MHz, CDCl 3 ): δ (m, 5H, H4, H2, H3, H5, H6 ), 7.36 (tt, 1H, H4, J=7.4, J=1.5), 7.19 (d, 1H, H6, J=1.0), 7.07 (d, 1H, H3, J=8.4), 5.53 (d, 1H, benzyl-h, J=14.0), 5.11 (dd, 1H, benzyl-h, J=14.0, J =9.7); 13 C MR (100 MHz, CDCl 3 ): δ (C2), (C1 ), (C5), (C3,C5 ), (C4), (C4 ), (C2,C6 ), ( J=2.0, C6), (C1), ( J=2.0, C3), ( J=2.6, benzyl-c); 31 MR (101 MHz, CDCl 3 ): δ Benzo[a] saligenyl chlorophosphite (7a) Quantities: 5a (1.08 g crude product), suspended in 65 ml ether, phosphorus(iii) chloride (0.63 ml, 7.17 mmol), pyridine (1.16 ml, 14.4 mmol), dissolved in 4.9 ml ether. Reaction periods: 10 min. after addition of phosphorus(iii) chloride, 3 h for the addition of the pyridine-ether solution, 2 h stirring at room temperature. This afforded racemic 7a as a crude yellow oil (850 mg, 100% yield: 1.48 g); The product showed degradation even in dry deuterated organic solvents, so it was only characterized by its phosphitylating properties in the next synthetic step. Benzo[b] saligenyl chlorophosphite (7b) Quantities: 5b (802 mg, 4.60 mmol), suspended in 65 ml International Medical ress

7 ovel cyclosal prodrugs of d4t ether, phosphorus(iii) chloride (0.47 ml, 5.32 mmol), pyridine (0.86 ml, 10.7 mmol), dissolved in 3.6 ml ether. Reaction periods: 15 min after addition of phosphorus(iii) chloride, 3 h for the addition of the pyridine ether solution, 2 h stirring at room temperature. This afforded racemic 7b as a crude slightly yellow solid (950 mg, 100% yield: 1.10 g); 1 H MR (400 MHz, CDCl 3 ): δ 7.78 (s, 1H, H6), 7.75 (s, 1H, H3), (m, 4H, H1, H2, H3, H4 ), (m, 1H, benzyl-h), (m, 1H, benzyl-h ); 13 C MR (100 MHz, CDCl 3 ): δ ( J=6.1, C2), (C4), (C5), (C4 ), (C1 ), (C2 ), (C3 ), ( J=2.0, C6), (C1), ( J=2.5, C3), ( J=2.5, benzyl-c); 31 MR (101 MHz, CDCl 3 ): δ Benzo[c] saligenyl chlorophosphite (7c) Quantities: 5c (800 mg, 4.59 mmol), dissolved in 28 ml ether, phosphorus(iii) chloride (0.47 ml, 5.32 mmol), pyridine (0.86 ml, 10.7 mmol), dissolved in 3.6 ml ether. Reaction periods: 5 min after addition of phosphorus(iii) chloride, 1.7 h for the addition of the pyridine ether solution, 2 h stirring at room temperature. This afforded racemic 7c as a crude slightly yellow solid (881 mg, 100% yield: 1.10 g); 1 H MR (400 MHz, CDCl 3 ): δ 7.85 (d, H4, J=8.1), 7.78 (d, H1, J=8.9), (m, 2H, H4, H3 ), (m, 1H, H2 ), 7.15 (d, 1H, H3, J=9.2), 5.71 (d, 1H, benzyl-h, J=12.0), 5.58 (d, 1H, benzyl-h, J=12.0); 13 C MR (100 MHz, CDCl 3 ): δ ( J=5.1, C2), (C6), (C4), (C1 ), (C5), (C3 ), (C2 ), (C4 ), (C3), ( J=14.8, C1), ( J=2.6, benzyl-c); 31 MR (101 MHz, CDCl 3 ): δ Chemical hydrolysis studies of the cyclosal phosphate triesters 300 µl of a 1.9 mm hydrolysis solution of the cyclosal nucleotide were prepared by dilution of a 50 mm DMS solution of the phosphate triester with DMS/water. To this hydrolysis solution, 5 µl of the internal standard AZT (5 mg in 500 µl water) were added. The reaction was started by addition of 300 µl of 50 mm phosphate buffer (B) previously warmed to 37 C, in order to reach the final concentrations (0.94 mm pronucleotide, 24.8 mm buffer salts). This solution was incubated at 37 C. To monitor the reaction, aliquots of 60 µl were taken from the hydrolysis mixture, poured into a small amount of glacial acetic acid and frozen in liquid nitrogen. These samples, including a starting sample directly taken after addition of B, were analysed by analytical HLC. For each chromatogram, the ratio of the integrals of the phosphate triester and AZT was calculated (standardized integration units=standardized IU) and plotted against the reaction time in hours. Calculation of exponential decay curves with commercially available software gave the half-life (t 1/2 ) of the prodrug compounds. For each compound, two determinations of t 1/2 were performed and the resulting values were averaged. Hydrolysis study in cell extracts Hydrolysis studies of the cyclosal phosphate triesters in human CEM/ cell extracts. 100 µl of human CEM/ cell extracts prepared according to a literature procedure (Saboulard et al., 1999) were mixed with 20 µl of a 70 mm aqueous magnesium chloride solution. The reaction was started by addition of 20 µl of a 1.5 mm solution of the cyclosal phosphate triester in DMS. Four separate samples of the above mentioned mixtures were incubated for 0, 2, 4 and 8 h, respectively. This was done to avoid contamination of the biological media by taking multiple samples from only one solution. All solutions were incubated at 37 C. To stop the reaction and for precipitation of protein, 300 µl acidified methanol (1 ml glacial acetic acid in 20 ml methanol) were added and the cap was kept at 0 C for 5 min. After centrifugation ( g, 10 min) and filtration of the supernatant, the sample was subject of HLC analysis (0 to 50 min TBAH-phosphoric acid buffer [0.55 mm, ph 3.8] with an acetonitrile gradient from 0 to 70%, flow 0.6 ml/min). Two separate samples for each time point and test compound were prepared and analysed. Determination of t 1/2 was performed analogously to that for the chemical hydrolysis studies except of using the absolute integral values instead of standardized IU. Identification of the hydrolysis products was based on the retention times of the reference compounds and co-elution experiments under identical analytical conditions. 31 -MR hydrolysis studies of the cyclosal phosphate triesters Approximately 7 µmol of the cyclosal-triesters were dissolved in 500 ml deuterated DMS and 500 µl of a 50 mm imidazole/hydrochloric acid buffer (ph 7.3). In the case of 3b, 300 µl deuterated DMS had to be added to afford a clear solution. The resulting kinetic solutions were transferred into a MR tube and investigated by 31 -MR spectroscopy (proton decoupled, 202 MHz, 256 scans each sample). All samples were stored at room temperature. Furthermore, some proton coupled 31 -MR spectra (202 MHz, 512 scans each sample) were recorded for the identification of the hydrolysis products. Materials and methods: virology Cells Human lymphocyte CEM cells were obtained from the American Tissue Culture Type Collection (ATCC) (Rockville, Md., USA). Antiviral Chemistry & Chemotherapy 13:1 7

8 C Ducho et al. Figure 2. Synthetic pathways to the target triesters 2a, b and 3a c H H (a) 61% (b) 37% H H 4b H 4a H (f) (f) H CH 3 (C) via 8 5a H H (f) X a, b; 7a c CI (g) 25-59% 2a, b; 3a c (f) (f) H 5b H H 5c H (d) 99% (e) 95% CH 3 H H (a) (i) (HCH) n, phenylboronic acid, C 2 H 5 CH, toluene, 110 C, 8 h; (ii) H 2 2 /H 2, THF, 0 C, 35 min. (b) 37% HCH/H 2, KH, 0 C to r.t., 24 h. (c) (i) MeH, conc. H 2 S 4,65 C, 20 h; (ii) LiAlH 4, THF, r.t. to 66 C, 3 h, no purification. (d) LiAlH 4, THF, r.t., 3 h. (e) 37% HCH/H 2, ah, 0 C, 1 h. (f) Cl 3, pyridine, Et 2, 20 C to r.t., 4 5 h, no purification. (g) (i) d4t 1, DIEA, CH 3 C or DMF/THF (2:1 v/v), 20 C to r.t., 1 h; (ii) TBH, 20 C to r.t., 1 h. Viruses HIV-1 (strain IIIB) was obtained from Dr RC Gallo and Dr M opovic (at that time at the ational Institutes of Health, Bethesda, Md., USA) and HIV-2 (strain RD) was obtained from Dr L Montagnier (at that time at the asteur Institute, aris, France) International Medical ress

9 ovel cyclosal prodrugs of d4t Antiviral assays The activity of the test compounds against HIV-1- and HIV-2-induced cytopathogenicity was examined in CEM cell cultures at days 4 5 post infection and the antiviral activity of the test compounds was estimated by microscopical examination of virus-induced giant cell formation. HIV-1 and HIV-2 were added at CCID 50 to the cell cultures (1 CCID 50 being the virus dose that is able to infect 50% of the cell cultures). Results The synthesis of the novel cyclosal phosphate triesters was performed in accordance to the established procedure (Meier et al., 1996, 1997b, 1998a): derivatives of salicyl alcohol 4, 5 were reacted with phosphorus(iii) chloride to yield saligenyl chlorophosphites 6, 7. These compounds were used as phosphitylating agents without further purification. Reaction of the chlorophosphites with d4t 1 gave phosphorus(iii) intermediates that were oxidized with t-butylhydroperoxide (TBH) to afford the target cyclosaltriesters 2, 3 in yields of 25 59% (Figure 2). The used salicyl alcohols 4a, b were not commercially available and had to be prepared as well as the ortho-hydroxymethylated naphthols 5a c. The simplest access to salicyl alcohols should be the reduction of appropriate carbonyl precursors. The commercially available carbonyl precursor of 5a, 1-hydroxy-2-naphthoic acid, had been esterified with methanol to afford ester 8 in 85% yield prior to reduction to allow mild reaction conditions for the preparation of the unstable compound 5a. Interestingly, also the corresponding chlorophosphite 7a turned out to be labile as indicated by its rapid decomposition in several deuterated organic solvents used for the preparation of the MR samples. Consequently, the yield in the subsequent synthesis of cyclosal compound 3a was the lowest of all triesters described (25%). Another elegant method for the preparation of salicyl alcohols is the direct hydroxymethylation with aqueous formaldehyde solution under basic conditions. This method has been employed for the synthesis of 4b and 5c (Blade-Font et al., 1982). Since this method usually is not very regioselective and no appropriate carbonyl precursor is available, the preparation of the 3-phenyl derivative 4a was performed in a different way. We chose a two-step protocol established by agata (agata et al., 1979) consisting of the preparation of a cyclic phenylboronic acid ester from 2-hydroxybiphenyl, paraformaldehyde, phenylboronic acid and propionic acid (catalyst), followed by oxidative cleavage of the cyclic boronic acid ester with hydrogen peroxide to afford 4a in 61% overall yield (Figure 2). All cyclosal phosphate triesters were hydrolyzed in phosphate buffer (B) at ph 7.3 and 37 C to estimate their chemical stability under physiological conditions. The 3-phenyl substituted compound 2a showed the highest half-life (t 1/2 =5.1 h) even in comparison to the unsubstituted cyclosal-d4tm prototype 9a (t 1/2 =4.4 h). Remarkably, the change of the phenyl substituent from 3- to 5-position resulted in a considerable loss in stability when the half-life dropped to 3.1 h. All benzo-annulated derivatives 3a c turned out to be even more labile. For benzo[a] and Table 1. Hydrolysis half-lives, product distributions and antiviral activity of the novel compounds 2, 3 and the reference compounds 1, 9 t 1/2 (h) EC 50 (µm) Amount of CEM/ CEM/ CEM/TK CC 50 (µm) Compound* (B) (CEM/) D (%) HIV-1 HIV-2 HIV-2 CEM/ 3-phenyl 2a ± ± ± ±5 5-phenyl 2b ± ± ± ±4 benzo[a] 3a 2.8 D ± ± ± ±17 benzo[b] 3b 1.4 D ± ± ± ±49 benzo[c] 3c 2.8 D ± ± ± ±3 unsubst. 9a 4.4 D ± ± methyl 9b ± ± ± methyl 9c 8.0** 9.2 D 0.18** 0.34** 0.18** 38** d4t ± ± ± ±32 *nly the substitution pattern in the cyclosal moiety is given. Hydrolysis half-life in phosphate buffer (B) at 37 C. Hydrolysis half-life in CEM/ cell extracts at 37 C. Amount of phenyl phosphate diester (D) 10 determined after 22 days by 31 -MR; remaining percentage represents d4tm amount. 50% effective concentration, or compound concentration required to inhibit HIV-induced giant cell (CEM) formation by 50%. 50% cytostatic concentration, or compound concentration required to inhibit CEM cell proliferation by 50%. **Data taken from Meier et al., 1998a. D, not determined. Antiviral Chemistry & Chemotherapy 13:1 9

10 C Ducho et al. benzo[c]-annulated compounds 3a, c half-lives of 2.8 h were determined, while 3b showed a half-life of 1.4 h (Table 1). Moreover, the phenyl-substituted prodrugs 2a, b were hydrolyzed in human CEM/ cell extracts to get insights into their intracellular fate. A slight decrease in stability was observed under these conditions in comparison to the experiments described above, but the order of stability (3-phenyl >5-phenyl) remained unchanged (Table 1). The subsequent investigation of the 3- and 5-methyl substituted reference compounds 9b, c in the cell extracts clearly demonstrated a general destabilizing effect of the phenyl substituents on the cyclosal moiety because they showed three- to fivefold higher half-lives. All hydrolysis studies described so far are based on the HLC analysis of samples taken from the hydrolysis mixtures. Hence, the identification of hydrolysis products has to be done by comparison of the chromatograms with the retention times of the potential cleavage products. Since neither d4t 1 nor thymine was detected in the cell extract experiments, enzymatic dephosphorylation and/or deglycosylation during the cleavage of the prodrugs was excluded. However, small amounts of hydrolysis products often cause problems in the quantification. 31 -MR spectroscopy offers significant advantages in terms of quantification and structure identification. It should be pointed out that the identification of the hydrolysis products in these studies is not exclusively based on their chemical shifts, but also on proton coupled 31 -MR experiments performed in some cases. Therefore, we carried out 31 -MR experiments in mixtures of DMS and imidazole/hydrochloric acid buffer solutions (Figures 3 and 4). Under these conditions, the cyclosal-triesters turned out to hydrolyze sufficiently slowly so that a precise MR monitoring of the hydrolytic reaction was possible. Based on our knowledge of typical chemical shifts for the expected products, further mechanistic conclusions could be drawn. The data obtained for the 3-phenyl derivative 2a are a good example for the high potential of the method. It shows the intermediate formation of a benzyl phosphate diester (steady state) and subsequently of d4tm and also for the first time traces of a phenyl phosphate diester 10 resulting from a spontaneous benzyl C- bond rupture (Figures 3 and 4, see also Figure 5). However, the intermediate benzyl phosphate diester was not observed during the hydrolysis of the benzo-annulated derivatives. The reason may be a very fast degradation of the intermediate diester to d4tm. Furthermore, the amount of the diester 10 relative to all products was determined after 22 days reaction period. While 2a and 3b only yielded small amounts of the undesired diesters 10 (maximum 3%), triester 2b gave no detectable amounts of diester 10. However, the benzo[a] and benzo[c] substituted cyclosal-d4tms 3a, c showed a Figure 3. (a) (b) Amount (%) high tendency to be cleaved in the wrong way to give diester 10 (Table 1, also see Figure 4). As expected, diester 10 proved to be chemically stable under the reaction conditions. Moreover, by integration of the triester resonance signal it was observed that the two diastereomers present in the sample did not hydrolyze with the same rate (Figures 3 and 4). This effect has been observed before in chemical hydrolysis studies of triester 9b (Meier et al., 1998a). There was a twofold difference detected in the hydrolysis rate. In the hydrolysis studies in B this difference cannot be detected due to non-resolution of the diastereomers. Finally, antiviral assays were carried out to determine the antiviral activity of the novel compounds. The 3-phenyl derivative 2a showed a threefold increase of activity against HIV-1 compared with d4t 1 and was as active as the unsubstituted reference compound 9a. More important was the complete retention of this antiviral activity in TK-defistart after 1 d after 2 d after 3 d after 7 d after 22 d 2a BD d4tm 2a (two diastereomers) D d4tm (ppm) phenyl phosphate diester Time (d) benzyl phosphate diester (a) Stack plot of the proton decoupled 31 -MR spectra obtained for the hydrolysis of 3-phenyl cyclosal d4tm 2a. (b) Quantitative evaluation of the hydrolysis process, obtained by integration of the appropriate MR-signals BD, benzyl phosphate diester; D, phenyl phosphate diester International Medical ress

11 ovel cyclosal prodrugs of d4t Figure 4. Stack plot of the proton decoupled 31 - MR spectra obtained for the hydrolysis of benzo[a]-cyclosal-d4tm 3a start after 1 d after 2 d after 3 d after 7 d after 22 d 11 cient cells. n the other hand, 2b and 3b not only turned out to be less effective against HIV-1 and -2 in wild-type CEM/ cells than 2a and 9a, but more importantly suffered from a significant loss of activity in the TK-deficient cell line. Although 3a, c showed good antiviral data in TKcompetent cells comparable to 2a, their activity decreased slightly in the TK-deficient cells, although less pronounced as observed for 2b and 3b. However, they were still more active as compared to d4t 1 in the CEM/TK cells. In spite of the use of biphenyl-based masking groups, compounds 2a, b did not possess increased cytotoxicity compared to 3-methyl-cycloSal-d4TM 9b (Table 1). Discussion d4tm D (ppm) D, phenyl phosphate diester 10. 3a (two diastereomers) The cyclosal moiety is cleaved due to the nucleophilic attack of a hydroxide anion on the phosphorus atom and subsequent development of the resonance stabilized phenolate. The resulting benzyl phosphate diester degrades spontaneously to the nucleoside monophosphate and the salicylic alcohol because of the different electronic properties of the ortho substituent (hydroxy-, a strong donor) compared with the starting compound (phosphate-, a very weak donor). Therefore, slightly basic ph conditions led to d4tm delivery from the cyclosal pronucleotides. The different hydrolysis pathway, the spontaneous cleavage of the benzyl ester bond yielding an inert phenyl phosphate diester, is normally not observed (Figure 5) (Meier et al., 1998a; Meier, 2002). Donor-substituted cyclosal derivatives of d4tm (X=3- methyl; 3,5-dimethyl) showed good antiviral activity due to their higher hydrolytic stability resulting from the poor stabilization of the primary hydrolysis product, the phenolate anion. articularly the high activity in TK deficient cells was a convincing proof for a successful TK bypass. n the contrary, acceptor-substituted cyclosal-d4tm triesters (X=5-Cl; 5-2 ) turned out to be of lower antiviral activity, a result of their lower hydrolysis stability due to a good stabilization of the phenolate anion (Meier et al., 1998a, 2002). As before, the hydrolysis half-lives determined for the pronucleotides in this work correlate to their chemical structures. For example, the higher stability of the 3-phenyl derivative 2a compared with the regioisomer 2b is a result of the more frozen position of the phenyl substituent. The bulky phosphate ester moiety in the ortho-position may disturb free rotation of the phenyl substituent (buttress effect). So, the overlapping of the p-orbitals in the two aromatic rings is poor, and a significant stabilization of the primary hydrolysis product (phenolate anion) by delocalizing the negative charge into the second ring may not be possible. n the other hand, the phenyl substituent in the 5-position should be able to rotate almost freely at room temperature, resulting in a higher orbital overlap and therefore in a higher tendency to stabilize and form the phenolate anion. More experimental validation has to be done to confirm this interpretation, for example, by temperaturedependent MR. Furthermore, the comparison of 2a, b to the reference 9a offers convincing insights. As the phenyl substituents should possess a very weak (+)I-effect as well as a (-)Meffect, they are expected to serve as weak electron withdrawing groups. Hence, a destabilization of the cyclosal nucleotides as observed in 2b should be the result. The slight stabilization of 2a compared with 9a can be related to two effects: first, the (-)M-effect of the phenyl substituent is lowered as a result of the poor orbital overlapping, leading to a dominance of the (+)I-effect and therefore a stabilization of the prodrug. Second, lipophilicity properties may play an important role as the phenyl group in 2a is positioned close to the region of the nucleophilic attack, the phosphate moiety. It is difficult to judge whether the first or the second effect is the key to the observed stabilization. The significant decrease in hydrolytic stability observed in the case of the benzo-annulated derivatives 3a c is a result of the excellent stabilization of the phenolate anion. The similar hydrolytic properties of 3a, c compared with the even more labile 3b can be related to the even better phenolate stabilization in the latter. As the half-lives determined in the cell extract experiments did not dramatically differ from those obtained from the kinetics in BS, a possible participation of enzymatic processes in the cleavage of the cyclosal compounds may be ruled out and pure hydrolytic cleavage is encountered. Furthermore, this is good evidence that the conditions used Antiviral Chemistry & Chemotherapy 13:1 11