Antiviral Research, 17 (1992) 197-212
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© 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-3542/92/$05.00 AVR 00530
Synthesis and anti-HIV activity of some novel lactyl and glycolyl phosphate derivatives Christopher McGuigan l, Caleb Nickson 2, Timothy J. O'Connor 3 and Derek Kinchington 3 IDepartment of Chemistry, University of Southampton, Highfield, Southampton, 2Department of Chemistry, University College London, London and 3Department of Virology, Medical College of St. Bartholomew's Hospital, Bartholomew Close, London, U.K. (Received 11 July 1991; accepted 7 August 1991)
Summary Novel phosphate triester derivatives of 3'-acetylthymidine, and of the antiHIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. These materials are designed to act as membrane-soluble pro-drugs of the bio-active free nucleotides. In particular, novel glycolate and lactate phosphate derivatives have been prepared. In vitro evaluation revealed the AZT compounds to have a pronounced and selective antiviral effect, the magnitude of which varied considerably with the nature of the phosphate blocking group. HIV; AZT; Nucleotide; Pro-drug; Lactate; Glycolate
Introduction Nucleoside analogues are firmly established as useful agents to combat viral infection and in the chemotherapy of cancer. However, they have a number of limitations, which might in part be overcome by the use of masked phosphate derivatives, if it is possible to release the bio-active free nucleotides intracellularly (Hunston et al., 1984; Chawla et al., 1984; Farquhar and Smith, 1985). We have applied this idea to the anti-herpetic agent araA (McGuigan et al., 1989) and the anti-cancer drug araC (Jones et al., 1989). Correspondence to." C. McGuigan, Dept. of Chemistry, University of Southampton, Highfield, Southampton, SO9 5NH, U.K.
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More recently, we have found that simple dialkyl phosphate derivatives of AZT, and other nucleoside analogues, are inactive as anti-HIV agents (McGuigan et al., 1990a), whereas trihaloethyl phosphates are active (McGuigan et al., 1990b). It was of interest to investigate whether other types of phosphate blocking groups were effective in this capacity. In this paper we disclose the observation that ester-containing phosphate protecting groups are particularly efficacious. We were prompted to investigate ester-containing groups by several factors. Firstly, we have noted that C-protected, N-linked amino acids are suitable blocking groups, leading to a significant anti-viral effect in many cases (Devine et al., 1990; McGuigan et al., 1990c). In the case of glycine compounds, the esters we will describe below are very closely related to these phosphoramidate analogues, with the link to the phosphate simply being altered from P-NH to P-O. Secondly, intracellular cleavage of the co-esters by carboxyl esterase would yield co-carboxylates. These may be trapped within the cell on the grounds of their increased polarity, and their phosphate hydrolysis may well be enhanced by intramolecular participation of the free carboxylate function. Lastly, it is noteworthy that ester containing groups are frequently used in pro-drug design, particularly as alcohol blocking groups (Bungdaard, 1985).
Materials and Methods
Commercially available Merck kieselgel F254 plates were used for the TLC and the components were visualised by UV light. Column chromatography was carried out using Woelm silica (32-63 mm) as the stationary phase. The ratio of silica-compound varied between 50:1 and 100:1 (w/w). EI MS was carried out by Dr. M. Mruzek on a VG 7070H mass spectrometer fitted with a Finnigan Incos II data system. FAB MS was carried out by the University of London mass spectrometry service on a VG Zab 1F spectrometer. Analytical HPLC was carried out on an ACS quaternary system, using a Spherisorb CN5~M column and an eluant of 5% water in MeCN, with a flow rate of 2 ml/min and detection by UV at 265 nm. Preparative HPLC was conducted on a Waters system using a Partisil 5 silica column and a mobile phase of 90% ethyl acetate/ 10% petroleum spirit, with a flow rate of 2 ml/min. Analytical data were obtained for solid products, courtesy of the team of Mr. A. Stones (University College London); oils and gums did not analyse within accepted limits, but were pure by spectroscopy, mass spectrometry, and HPLC. Phosphorus-31 N M R spectra of phosphorylating reagents were recorded on a Varian XL-200 spectrometer operating at 82 MHz. Phosphorus-31 NMR spectra of phosphate triesters were recorded on a VXR-400 spectrometer operating at 164 MHz and are reported in units of 6 relative to 85% phosphoric acid as external standard, positive shifts are downfield. Carbon-13 NMR spectra were recorded on a Varian XL-200 spectrometer operating at 50 MHz for all compounds except nucleotides, which were recorded on a Varian VXR-400 spectrometer operating
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at 100 MHz and are reported in units of ~ relative to CDC13 at 77000 ppm. Both phosphorus-31 and carbon-13 N M R spectra were proton noise decoupled and all signals were singlets unless otherwise stated. H-1 N M R spectra were recorded on a Varian XL-200 spectrometer operating at 200 MHz for all nonnucleoside containing compounds which were analysed with a Varian VXR-400 spectrometer operating at 400 MHz and are reported in units of 6 relative to internal CHC13 at 7.240 ppm unless otherwise stated. All N M R spectra were recorded in CDC13. All experiments involving water-sensitive reagents were carried out under scrupulously dry conditions. Where needed, anhydrous solvents and reagents were obtained in the following ways: pyridine was heated under reflux over calcium hydride for several hours, distilled and stored over activated molecular sieves. N-methylimidazole was heated to 100°C over Call2 for 4 h, then distilled under reduced pressure. Tetrahydrofuran was refluxed over potassium for several hours, distilled and stored over activated molecular sieves. Phosphoryl chloride was distilled prior to use.
Ethyl (ethyl glycolyl) phosphorochloridate ( la) Ethyl glycolate (2.91 g, 2.91 ml, 0.03 mol) and triethylamine (2.64 g, 3.64 ml, 0.03 mol), in diethyl ether (100 ml) were added dropwise over 1 h to a vigorously stirred solution of ethyl phosphorodichloridate (4.68 g, 3.41 ml, 0.03 mol) in diethyl ether at -60°C. After stirring for 4 h at this temperature the reaction mixture was allowed to warm to room temperature and left stirring overnight. Filtration and concentration under reduced pressure, followed by hexane (ca 50 ml) extraction afforded an oil (4.75 g, 79%). 6p 3.343.
Propyl (ethyl glycolyl) phosphorochloridate (lb) This was prepared by a method analogous to that used for (la) above. Thus, from 2.91 g (0.03 mol) of ethyl glycolate was isolated 4.03 g (62%) of (lb). fie 3.163.
Hexyl (ethyl glycolyl) phosphorochloridate (lc) This was prepared by a method analogous to that used for (la) above. Thus, from 0.24 g (2 mmol) of ethyl glycolate was isolated 0.6 g (92%) of (lc). fie 3.841; 6H 4.725 (2H, d, C(O)CH20, J=4.1 Hz), 4.27 (4H, m, CH2OC(O), CH2CH_H_20), 1.754 (2H, quintet CH2CH2CH20, J=7.25 Hz), 1.32 (9H, m, CHsCH2OC , CH3(CH___2)2CH2CH2CH20), 0.902 (3H, t, C H3CH2CH2, J = 7.14 Hz); found C 42.30%, H 7.05, P 10.68, C1 11.88; CloH2oOsPCI requires C 41.89%, H 7.03, P 10.80, C1 12.37.
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Dodecyl (ethyl glycolyl) phosphorochloridate (ld) This was prepared by a method analogous to that used for (la) above, except that an initial reaction temperature of -30°C was used. Thus, from 2.0 g (0.02 mol) of ethyl glycolate was isolated 7.1 g (96%) of (ld). 6c 166.393 (d, C(O), J = 8.1 Hz), 70.575 (d, CHzCH20, J=7.7 Hz), 63.899 (d, C(O)CH2OP, J = 5.9 Hz), 61.841 (CH2OC), 31.806 (_CHzCH2CH20), 29.512-22.586 ([_C__H2]s,m), 22.586 (CH3CH2CH2), 14.010 (_CH3CH20), 13.920(CH3R); 6p 3.577; m/e (EI) 373 (MH +, ~C1, 1.47%), 371.1754 (MH +, C16H33OsPC1 requires 371.1754, 5.00), 335 (M + -C1, 0.92), 271 (M + -CH3[CH2]6, 8.14), 205 (MH2 + - C H 3 [CH2]ll, 37C1, 35.43), 203 (MH2 + - CH3[CH2]ll, 100), 111 ([CHz]vCH +, 4.33), 83 ([CHz]sCH +, 10.14), 69 ([CHz]4CH +, 18.40), 55 ([CHz]3CH +, 33.28), 41 ([CHz]2CH +, 47.13).
3'-O-Acetylthymidine-5'-ethyl (ethyl glycolyl) phosphate (2a) To a stirred solution of 3'-O-acetylthymidine (0.61 g, 0.002 mol), in pyridine (10 ml) was added phosphorochloridate (la) (1.38 g, 1.27 ml, 0.06 mol). After leaving for 96 h., the reaction mixture was concentrated to dryness and triturated with toluene (ca 3 x 10 ml). Purification was achieved by flash column, chromatography on silica (ca 100 g) eluted with 5% methanol in chloroform. Pooling and evaporation of appropriate fractions afforded a colourless gum (0.22 g, 24%). 6c 170.389 (CH3C(O), A), 170.368 (CH3C(O), B), 167.862 (d, C(O)CH2, J=4.3 Hz, B), 167.608 (d, C(O)CH2, J=4.7 Hz, A), 163.927 (C-2, A), 163.926 (C-2, B) 150.666 (C-4, A), 150.650 (C-4, B), 135.026 (C-6, A), 134.926 (C-6, B), 111.612 (C-5, A), 111.562 (C-5, B), 84.317 (C-I', B), 84.242 (C-I', A), 82.637 (d, C-4', J=8.0 Hz, B), 82.554 (d, C-4', J=8.2 Hz, A), 74.484 (C-3', B), 74.464 (C-3', A), 67.257 (d, C-5', J = 5.2 Hz, B), 67.190 (d, C5', J=5.7 Hz, A), 64.758 (d, C(O)C__H2OP, J=5.3 Hz, B), 64.699 (d, C(O)CH2OP, J=6.0 Hz, A), 63.850 (d, CH3CH2OP, J--5.3 Hz, B), 63.660 (d, CH3CHzOP, J = 7.0 Hz, A), 61.648 (CH3CH2OC, A), 61.590 (CH3CH2OC, B), 36.993 (C-2', B), 36.931 (C-2', A), 20.776 (CH3C(O)), 15.891 (m, CH3CHzOP), 13.929 (CH3CH2OC), 12.402(5-Me); 6p 1.723, 1.548 (A:B; 2:1); 6/-/9.897 (1H, s, NH), 7.476, 7.458 (1H, d, H-6, J-- 1.22, 1.17 Hz), 6.36 (1H, m, H-I'), 5.27 (1H, m, H-3'), 4.57 (2H, m, C(O)CH20), 4.36 (2H, m, H-5'), 4.2294.005 (5H, m, CH3CH2OC, H-4', CH3CH_zOP), 2.35 (1H, m, H-2'), 2.16 (1H, m, H-2'), 2.052 (1H, s, CH3C(O)), 1.869, 1.872 (3H, d, base CH3, J = 1.09, 1.13 Hz), 1.37 (3H, m, CH3CH2OP), 1.218 (3H, t, CH__3CH2OC, J=7.68 Hz).
3'-O-A cetylthymidine-5'-(propyl ethyl glycolyl) phosphate (2b) This was prepared by a method entirely analogous to that used for (2a) above, except that the reaction was stirred for 70 h, and the chromatographic column was eluted much more slowly (0.5% methanol in chloroform), allowing a partial separation of the diastereoisomers; the pure diastereoisomers being
201 isolated as oils by preparative HPLC. Thus, from 0.54 g of 3'-0acetylthymidine was isolated 0.31 g (32%) of (2b).
Fast isomer (0.16 g, 16%). 6c 170.389 (CH3C(O)), 167.736 (d, C(O)CH2, J=4.8 Hz), 163.554 (C-2), 150.450 (C-4), 135.154 (C-6), 111.754 (C-5), 84.394 (C-I'), 82.794 (C-4', J=8.4 Hz), 74.628 (C-3'), 70.091 (d, CH2CH20, J=6.1 Hz), 67.321 (d, C-5', J = 5.7 Hz), 63.666 (d, C(O)CHzOP, J=5.0 Hz), 61.734 (CH3CH2OC), 37.089 (C-2'), 23.572 (d, CH2CH2OP, J=7.1 Hz), 20.896 (CH3C(O)), 14.073 (_CH3CH2OC), 12.329 (base CH3), 9.868 (_QH3CHzCH20); 6,0 0.776; 6/¢ 8.836 (1H, s, NH), 7.483 (1H, d, H-6, J = 1.26 Hz), 6.358 (1H, q, H-I'), 5.257 (1H, d, H-3', J = 5.98 Hz), 4.56 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.170 (2H, q, CH3CH__2OC, J=6.56 Hz), 4.12 (1H, m, H-4'), 4.05 (2H, m, CHzCH__20), 2.34 (1H, m, H-2'), 2.22 (1H, m, H-2'), 2.091 (3H, s, CH3C(O)), 1.877 (3H, d, base CH3, J = 1.01 Hz), 1.674 (2H, sextet, CH3CHHzCHzO, J = 6.77 Hz), 1.218 (3H, t, CH3CHzOC, J=7.32 Hz), 0.901 (3H, t, CH3CH2CH2, J = 7.36 Hz); m/e (FAB) 493 (MH +, 0.05%), 492 (M +, 0.01), 102 (C4H603 +,
lOO). Slow isomer (0.15 g, 16%). 6c 170.409 (CH3__C(O)), 167.719 (d, C(O)CH20, J=4.6 Hz), 163.715 (C-2), 150.378 (C-4), 135.249 (C-6), 111.687 (C-5), 84.561 (C-I'), 82.833 (d, C-4', J = 8.3 Hz), 74.589 (C-3'), 70.187 (d, CH2CH20, J = 5.9 Hz), 67.374 (d, C-5' J=6.0 Hz), 63.753 (d, C(O)CH2, J=5.4 Hz), 61.753 (_C_H2OC), 37.198 (C-2'), 23.540 (d, CH2CH20, J = 6.8 Hz), 20.896 (_CH3C(O)), 14.076 (C_H3CH20), 12.294 (base CH3), 9.866 (_C_H3CH2CH2); 6e 0.362; 5z4 8.879 (1H, s, NH), 7.463 (1H, d, H-6, J = 1.22 Hz), 6.357 (1H, q, H-I', J = 6.78 Hz), 5.282 (1H, d, H-3', JH-4'= 5.02 Hz), 4.56 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.183 (2H, q, CH3CH_2OC, J = 6.71 Hz), 4.12 (1H, m, H-4'), 4.05 (2H, m, CH2CH2OP), 2.34 (1H, m, H-2'), 2.22 (1H, m, H-2'), 2.091 (3H, s, CH3C(O)), 1.878 (3H, d, base CH3, J = 0.98 Hz), 1.668 (2H, sextet, CH3CH_zCH20, J = 6.69 Hz), 1.224 (3H, t, CH3CH2OC, J = 7.30 Hz), 0.895 (3H, t, CH3CH2OP, J = 7.36 Hz); m/e (FAB) 493 (MH +, 0.01%), 492 (M +, 0.01), 73 (C2H5OCO +, 100). 3'-O-Acetylthymidine-5'-hexyl (ethyl glycolyl) phosphate (2c) This was prepared by a method entirely analogous to that used for (2b) above, except that the chromatographic column was eluted with 0.55% methanol in chloroform, which gave a near-complete separation of the diastereoisomers. HPLC indicated the 'fast' isomer to be pure, whilst the 'slow' isomer contained 3% of the other diastereomer. Thus, from 0.67 g of 3'-0acetylthymidine was isolated 0.66 g (58%) of (2c) as a solid.
Fast Isomer (0.36 g, 32%). 6c 170.470 (CH3C(O)), 167.69 (d, C(O)CH2, J=4.9 Hz), 163.735 (C-2), 150.564 (C-4), 135.041 (C-6), 111.778 (C-5), 84.495 (C-I'), 82.854 (d, C-4', J = 8.2 Hz), 74.629 (C-3'), 68.882 (d, CHzCH2OP, J = 6.1 Hz), 67.340 (d, C-5', J = 5.2 Hz), 63.702 (C(O)CH2OP), 61.731 (CH3CH2OC),
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37.175 (C-2'), 30.173 (d, CH2CH2OP, J=6.1 Hz), 29.688 (_C_HzCH2CH20), 25.605 (CH3CHzCH2), 22.464 (CH3C_H2CH2), 20.887 (CH3C(O)), 14.099 (C_H3CH2OC), 13.928 (_C_H3CHzCH2), 12.232 (base CH3); fie 1.782; 6,4 9.009 (1H, broad s, NH), 7.502 (1H, d, H-6, J = 1.30 Hz), 6.42 (1H, m, H-I'), 5.31 (1H, m, H-3'), 4.62 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.23 (2H, m, CH3CH__2OC), 4.18 (1H, m, H-4'), 4.15 (2H, m, CHzCH2OP), 2.40 (1H, m, H2'), 2.38 (1H, m, H-2'), 2.07 (3H, s, CH3C(O)), 1.932 (3H, s, base CH3), 1.69 (2H, q, CH2CHzCH20, J=6.65 Hz), 1.25 (I1H, m, CH3CH2OC, (CH2)4), 0.928 (3H, t, CH__3CHzCH2, J=7.67 Hz); m/e (FAB) 536 (MH2 +, 0.5%), 535 (MH +, 6), 433 (3), 267 (acetylthymidine. H + - H 2 0 , 5), 147 (C6H1202 p+, 100), 81 (C5H50 +, 45).
Slow isomer (0.30 g, 26%). 6c 170.518 (CH3C(O)), 167.708 (d, C(O)CH2, J=4.9 Hz), 163.835 (C-2), 150.624 (C-4), 135.081 (C-6), 111.748 (C-5), 84.361 (C-I'), 82.757 (d, C-4', J = 8.2 Hz), 74.624 (C-3'), 68.240 (d, CH2CHzOP , J = 6.1 Hz), 67.331 (d, C-5', J = 5.2 Hz), 63.733 (C(O)CHzOP), 61.726 (CH3CH2OC), 37.124 (C-2'), 30.173 (d, CH2CHzOP J=6.1 Hz), 29.633 (_C_HzCH2CH20), 25.835 (CH3CH2CH2), 22.962 (CH3CHzCH2), 20.826 (_C_H3C(O)), 14.048 (_C_H3CH2OC), 13.238 QC_H3CH2CH2), 12.312 (base CH3); fie 1.625; 6/-/9.282 (1H, broad s, NH), 7.535 (1H, d, H-6, J = 1.30 Hz), 6.32 (1H, m, H-I'), 5.323 (1H, m, H-3'), 4.61 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.23 (2H, m, CH3CH2OC), 4.17 (1H, m, H-4'), 4.15 (2H, m, CH2CHzOP), 2.38 (1H, m, H2'), 2.26 (1H, m, H-2'), 2.07 (3H, s, CH3C(O)), 1.902 (3H, d, base CH3, J = 1.16 Hz), 1.66 (2H, m, CH2CH2CH20) 1.24 (11H, m, CH3CH2OC, (CH2)4), 0.902 (3H, t, CH3CHzCH2, J=7.67 Hz); m/e (FAB) 536 (MHz +, 1.0%), 535 (MH + 8), 433 (9), 267 (acetylthymidine. H + --H20, 6), 149 (C6H1402 p+, 100), 8; (C5H50 +, 78); found C 49.70%, H 6.91, N 4.79, P 5.01; CzzH35NzOllP requires C 49.44, H 6.60, N 5.24, P 5.79. 3'-Azido-3'-deoxythymidine-5'-propyl (ethyl glycolyl) phosphate (3a) Phosphorochloridate (lb) (1.10 g, 0.004 mol) was added to AZT (0.2 g, 0.75 mmol) in pyridine (10 ml) with stirring. After leaving for 14 days, water (ca 2 ml) was added and the reaction mixture concentrated to dryness and triturated with toluene (3 × 20 ml), Purification by flash column chromatography (silica ca 60 g) eluted with 3% methanol in chloroform gave a solid (0.13 g, 37%); 6c 167.785 (d, C(O)CH2 J = 4.4 Hz), 167.716 (d, C(O)CH2, J = 4.8 Hz), 163.913 (C2), 150.392 (C-4), 135.191 (C-6), 135.073 (C-6), 111.395 (C-5) 111.352 (C-5), 84.516 (C-I'), 84.469 (C-I'), 82.144 (d, C-4', J=8.3 Hz), 70.128 (d, CHzCH20, J=6.6 Hz), 70.062 (d, CHzCH20, J=6.5 Hz), 66.573 (d, C-5', J=5.8 Hz), 66.440 (d, C-5', J=5.3 Hz), 63.717 (d, C(O)CH2, J=4.3 Hz), 63.639 (d, C(O)CH2 J = 5.1 Hz), 61.683 (_C_H2OC), 60.110 (C-3'), 60.051 (C-3'), 37.395 (C2'), 37.326 (C-2'), 23.415 (d, CH3CH2CH2, J=7.0 Hz), 13.971 (CH3CH20), 12.290 (base CH3), 12.263 (base CH3), 9.767 (_C_H3CH2CH2); 6p 0.766, 0.362 (A:B, 1:1); 6/~ 9.732 (1H, s, NH), 7.394, 7.379 (1H, d, H-6, J = 1.26, 1.19 Hz),
203 6.21 (1H, m, H-I'), 4.58 (2H, m, C(O)CH2) 4.39 (1H, m, H-3'), 4.34 (2H, m, H5'), 4.18 (2H, m, CHH_2OP),4.04 (2H, m, CH2OC), 3.98 (1H, m, H-4'), 2.43 (2H, m, H-2'), 1.863, 1.855 (3H, d, base CH3, J--1.07, 1.07 Hz), 1.65 (2H, m, CHaCH__2CH2) , 1.226 (3H, t, C H 3 C H 2 0 , J=7.17 Hz), 0.899 (3H, t, C H a C H E C H 2 , J=7.36 Hz); m/e (EI) 476 (MH + 5.91%), 373 (1.01), 350 (MH +-thymine, 1.85), 329 (AZTPOeH +, 0.98), 307 (MH2 +-thymine - Nail, 36.90), 250 (AZT.H + - - H 2 0 , 17.19), 209 (M+-AZT, 4.36), 185 (18.41) 167 (PrO[EtO]PO2, 14.56), 139 (PrOPOaH, 54.71), 127 (thymine H +, 23.29), 126 (thymine +, 30.36), 123 (PrOPO2H, 18.71), 81 (C5H50 +, 100); found C 42.80%, H 5.39, N 14.25, P 6.45; C17H2609N5 P requires C 42.95, H 5.51, N 14.73, P 6.52.
3'-Azido-3'-deoxythymidine-5'-dodecyl (ethyl glycolyl) phosphate (3b) Phosphorochloridate (ld) (1.66 g, 0.004 mol), was added to AZT (0.2 g, 0.007 mol) and N-methylimidazole (0.49 g, 0.48 ml, 0.006 mol) in tetrahydrofuran (2.5 ml) at room temperature. After five min a further 10 ml of tetrahydrofuran was added to the solidified mixture. After leaving for two days the reaction mixture was concentrated to dryness under reduced pressure, dissolved in chloroform (35 ml) and saturated sodium bicarbonate solution (15 ml) was added. The emulsion was concentrated to dryness and dissolved in chloroform (ca 4 ml). This was added portionwise to a stirred solution of petroleum ether (b.p. 30-40°C) (ca 500 ml) and the mixture refrigerated overnight at - 20°C. Further purification of the product was achieved by flash column chromatography (silica ca 60 g) eluted with 4% methanol in chloroform, pooling and evaporating the appropriate fractions. Final purification was achieved by flash chromatography (silica ca 60 g) eluted with chloroform. Pooling and evaporating the appropriate fractions gave the product as a solid (0.36 g, 79%). 6c 167.793 (d, C(O)CH2, A, J=4.3 Hz), 167.717 (d, C(O)CH2, B, J=4.6 Hz), 163.855 (C-2, A), 163.838 (C-2, B), 150.391 (C-4), 135.155 (C-6, A), 135.033 (C-6, B), 111.425 (C-5, A), 111.382 (C5, B), 84.509 (C-I', A), 84.453 (C-I', B), 82.166 (d, C-4', J=8.1 Hz), 68.755 (d, C H z C H z C H 2 0 , A, J=6.1 Hz), 68.678 (d, CHzCH2CH20, B, J=5.5 Hz), 66.564 (d, C-5', A, J=5.9 Hz), 66.444 (d, C-5', B, J = 5.3 Hz), 63.723 (d, C(O)CH2, A, J = 5.2 Hz), 63.643 (d, C(O)CHz, B, J = 5.1 Hz), 61.682 (_C_H2OC), 60.124 (C-3', A), 60.066 (C-3', B), 37.435 (C-2', A), 37.356 (C-2', B), 31.771 (_CH2CHzCH20), 30.059 (d, CH2CH2CH20, J=6.9 Hz), 29.486-25.220 (m, (_~_H2)8), 22.555 (CH3CHzCH2), 13.999 (C_H3CH20), 13.985 (_C_H3R), 12.320 (5Me, A), 12.293 (5-Me, B); 6p 0.510, 0.137 (A:B, 7:3); 6/_/9.7 (1H, s, NH), 7.428, 7.411 (d, 1H, H-6, A, B, J=1.22, 1.19 Hz), 6.253, 6.250 (1H, t, H-I', A, B, J=7.31, 6.95 Hz), 4.60 (2H, m, C ( O ) C H 2 ) , 4.42 (1H, m, H-3'), 4.36 (2H, m, H5'), 4.22 (2H, m, CH2OP), 4.102 (2H, q, CH2OC, J=6.9 Hz), 4.01 (1H, m, H4'), 2.38 (1H, m, H-2'), 2.31 (1H, m, H-2'), 1.895, 1.888 (3H, d, 5-Me, B, A, J = 1.01, 0.95 Hz), 1.66 (2H, m, CHzCH20), 1.26 (3H, m, CH3CH20), 1.215 (18H, m, ( C H 2 ) 9 ) , 0.838 (3H, t, CH3CHzCH2, J = 6.68 Hz); m/e (El) 433 (MH +
204
-C12H25, 26.79%), 250 (AZT.H + - H 2 0 , 18.25), 186 (C12H25OH +, 6.87), 185
(C12H25O+, 78.68), 167 (C4HyO3PO2H +, 13.34), 127 (30.11), 126 (thymine.H +, 49.14), 113 (127 -CH2, 18.63), 99 (113 -CH2, 22.85), 85 (99CH2, 11.73), 81 (C5H50 +, 100), 71 (85 -CH2, 27.54), 57 (71 -CH2, 64.23), 43 (57 --CH2, 85.41), 29 (C2H5+, 80.26); m/e (FAB) 603 (MH2 +, 2%), 602 (MH +, 8), 434 (MH2 + --C12H25, 0.5), 433 (MH + --C12H25, 1), 353 (M + + O H - A Z T , 1), 250 (AZT.H + -H20, 8), 185 (C12H250+, 11), 81 (C5H50 +, 100); found C 51.49%, H 7.41, N 11.54, P 5.19; C26H44N509P requires C 51.91, H 7.37, N 11.64, P 5.15.
Methyl lactyl phosphorodichloridate (S)-( - )-Methyl lactate (8.72 g, 8.00 ml, 0.08 mol) and triethylamine (8.46 g, 11.6 ml, 0.08 mol) in diethyl ether (200 ml) were added to a vigorously stirred solution of phosphoryl chloride (12.9 g, 7.83 ml, 0.08 mol) in diethyl ether (200 ml) at -78°C. The reaction was kept at this temperature for a further 12 h with stirring. After warming to room temperature, filtration and concentration under reduced pressure gave an oil that was distilled (67-70°C/0.05 mm Hg) to afford the product as a colourless oil (12.57 g, 68%). 6c 168.405 (d, C(O), J=6.0 Hz), 75.309 (d, CH, J=8.8 Hz), 52.747 (CH30), 18.543 (d, CH3CH, J=6.7 Hz); 6p 6.458; 61-/5.12 (1H, m, CH), 3.759 (3H, s, CH30), 1.629 (3H, d x d , CH3C, Jp= 1.05 Hz, JcH=6.8 Hz); found C 21.66%, H 3.19, P 15.01; C4H704PC12 requires C 21.74, H 3.19, P 14.02.
Methyl (methyl laetyl) phosphorochloridate (4a) This was prepared by a method entirely analogous to that used for methyl lactyl phosphorodichloridate above, except that a reaction temperature of -78°C was used for 12 h, followed by 14 h at -40°C. Thus, from 2.2 g of methyl lactyl phosphorodichloridate was produced 2.03 g (94%) of crude (4a). 6c 169.325 (d, C(O), J=5.4 Hz), 169.095 (d, C(O), J=6.2 Hz), 73.179 (d, CH, J=2.3 Hz), 73.051 (d, CH, J=2.0 Hz), 55.601 (d, CH3OP, J--7.1 Hz), 52.366 (CH3OC), 18.611 (d, CH3CH, J=5.4 Hz), 18.392 (d, CH3CH, J=6.2 Hz); 6e 4.001, 3.217 (A:B, 1:1); m/e (EI) 219 (MH +, 37C1, 0.07%), 217.005 (MH +, CsHIIOsPC1 requires 217.003, 0.39), 185 (MH + -CH3OH, 3.34), 181 (M + -C1, 13.72), 157 (MH + -C2H402, 100), 133 (CH3OPO2HzC1 + , 37 C1, 21.03), 131 (CH3OPO2HzC1 +, 67.74), 115 (CH3OPOCI +, 37C1, 8.60), 113 (CH3 OPOC1 +, 25.73); found C 26.86%, H 4.56, P 14.09; CsH10POsC1 requires C 27.73, H 4.65, P 14.30.
Propyl (methyl lactyl) phosphorochloridate (4b) This was prepared by a method entirely analogous to that used for (4a) above, except that a reaction temperature of -60°C was used for 4 h, followed by 14 h at ambient temperature. Thus, from 4.369 of (S)-( - )-methyl lactate
205 was produced 6.36 g (62%) of (4b) (b.p. 128°C/0.01 mmHg). 6c 169.590 (d, C(O), J=5.3 Hz), 169.400 (d, C(O), J=6.5 Hz), 73.121 (d, CH, J=7.7 Hz), 72.997 (d, CH, J=7.6 Hz), 71.730 (d, CH20, J=7.7 Hz), 52.502 (CH30), 52.467 (CH30), 22.968 (d, CHzCH20, J = 8.1 Hz), 22.940 (d, CH2CH20, J = 7.8 Hz), 18.846 (d, CH3CH, J=5.5 Hz), 18.573 (d, CH3CH, J=6.0 Hz), 9.629 (_C_H3CH2); fie 4.553, 3.644 (A:B, 9:11); m/e (EI) 247 (MH +, 37C1, 0.04%), 245.0345 (MH +, C7H15OsPC1 requires 245.0346, 0.43), 205 (MH + -C3H6, 37C1, 2.30), 203 (MH + -C3H6, 7.49), 187 (M + --C2H302, 37C1, 12.79), 185 IM + -C2H302, 37.69), 167 (MH + - C 3 H 7 - C I , 17.32), 145 (M + -C4H603, C1, 49.39), 143 (M + --C4H603, 100), 87 (C4H702+, 35.42), 59 (C2H302 +, 22.61).
3'-Azido-3'-deoxythymidine-5'-methyl (methyl lactyl) phosphate (5a) This was prepared by a method entirely analogous to that used for (3b) above, except that 1% methanol in chloroform was used as the chromatographic eluant. Thus, from 0.08 g of AZT was isolated 0.13 g (73%) of (5a) as a gum. 6c 170.604 (m, C(O)CH), 163.593 (C-2, A), 163.546 (C-2, B), 150.134 (C4, A), 150.120 (C-4, B), 135.179 (C-6, A), 135.136 (C-6, B), 111.607 (C-5, B), 111.484 (C-5, A), 84.621 (C-I'), 82.321 (d, C-4', A, J=8.3 Hz), 81.971 (d, C4', B, J=7.8 Hz), 72.530 (d, CHO, A, J=5.3 Hz), 72.370 (d, CHO, B, J=5.1 Hz), 66.738 (d, C-5', A, J=5.8 Hz), 66.331 (d, C-5', B, J=5.6 Hz), 60.141 (C-3', A), 59.902 (C-3', B), 55.045 (d, CH3OP, B, J= 11.8 Hz), 54.409 (d, CH3OP, A, J= 11.9 Hz), 52.719 (_c_n3oc, A), 52.665 (_C_H3OC,B), 37.605 (C-2', A), 37.489 (C-2', B), 19.082 (m, CH3CH), 12.369 (5-Me); 6p -2.442, -2.487 (A:B, 2:3); f n 8.95 (1H, s, NH), 7.426, 7.399 (1H, d, H-6, A, B, J = 1.25, 1.22 Hz), 6.24 (1H, m, H-I'), 4.97, 4.91 (1H, m, CHO, A, B), 4.523-4.245 (3H, m, H-3', H-5'), 4.02 (1H, m, H-4'), 3.877-3.731 (6H, m, CH3OP, CH3OC), 2.42 (1H, m, H-2'), 2.31 (1H, m, H-2'), 1.922, 1.916 (3H, d, 5-Me, B, A, J= 1.18, 1.22 Hz), 1.56 (3H, m, CH_H_H_~CH).
3'-Azido-3'-deoxythymidine-5'-propyl (methyl lactyl) phosphate (5b) This was prepared by a method entirely analogous to that used for (5a) above. Thus, from 0.13 g of AZT was isolated 0.33 g (73%) of (5b) as a gum. 6c 170.866 (d, C(O)CH, A, J=3.7 Hz), 170.511 (d, __C(O)CH, B, J=4.6 Hz), 163.953 (C-2, A), 163.909 (C-2, B), 150.416 (C-4, A), 150.382 (C-4, B), 135.029 (C-6, A), 134.995 (C-6, B), 111.464 (C-5, B), 111.329 (C-5, A), 84.440 (C-l'), 82.176 (d, C-4', A, J=8.2 Hz), 81.971 (d, C4', B, J=8.0 Hz), 72.205 (d, CHO, A, J= 5.2 Hz), 72.052 (d, CHO, B, J= 5.2 Hz), 70.160 (d, CH2CH20, B, J=6.1 Hz), 69.659 (d, CH2CH20, A, J=5.2 Hz), 66.566 (d, C-5', A, J=6.1 Hz), 66.185 (d, C-5', B, J=5.6 Hz), 60.160 (C-3', A), 59.909 (C-3', B), 52.514 (CH30, A), 52.441 ( c n 3 0 , B), 37.356 (C-2', A), 37.238 (C-2', B), 23.361 (m, CH2CH20), 19.972 (d, __CH3CH,B, J=5.4 Hz), 18.918 (d, CH3CH, A, J=5.6 Hz), 12.259 (5-Me), 9.765 (_C_H3CH2, A), 9.732 (_C_H3CH2, B); 6p -2.168,
206 -2.312 (A:B 4:5); 6/4 10.078, 10.030 (1H, s, NH, B, A), 7.43 (1H, M, H-6), 6.25 (1H, m, H-I'), 4.94 (1H, m, CHO), 4.36 (1H, m, H-3'), 4.32 (2H, m, H-5'), 4.103 (1H, q, H-4', J = 6.88 Hz), 4.02 (2H, m, CH2CH20), 3.739 (3H, s, CH30), 2.42 (1H, m, H-2'), 2.34 (1H, m, H-2'), 1.903, 1.896 (3H, d, 5-Me, B, A, J = 1.23 Hz, J = 1.19 Hz), 1.689 (2H, septet, CH2CH20, J = 7.01 Hz), 1.53 (3H, m, CH3CH), 0.94 (3H, m, CH3CH2); m/e (EI) 476 (MH +, 0.40%), 416 (M + -C2H302, 0.01), 373 (MH + -C4H703, 0.11), 350 (M + -thymine, 0.12), 329 (1.19), 307 (9.20), 250 (AZT.H + - H 2 0 , 2.48), 227 (1.64), 209 (MH + - A Z T ) , 185 (C4HvO3PO3H3 +, 3.44), 167 (C4HvO3PO2H +, 11.46), 126 (thymine.H +, 7.99), 125 (thymine +, 16.13), 87 (C4H702+, 3.52), 99 (C5H702+, 11.42), 81 (C5H50 +, 100).
Biological evaluation C8166 T-lymphoblastoid cells were incubated with 10 TCIDso HIV-1 (RF strain) at 37°C for 1.5 h. The cells were washed three times with phosphatebuffered saline (PBSA:Dulbecco A); aliquots of 2 × 105 cells were resuspended in 1.5 ml of growth medium in the presence of each compound at half log dilutions and incubated at 37°C for 72 h in a 95:5% air/CO2 atmosphere. HIV antigen was measured in the supernatants using a commercial ELISA following the steps described by the manufacturers (Coulter Electronics Ltd., Luton, Beds, U.K.) (Kinchington et al., 1989). AZT and ddC were used as internal controls together with uninfected and infected, untreated cells. To test for compound toxicity, aliquots of 2 × 105 uninfected cells were cultured with the compounds for 72 h. The cells were then washed with PBSA and resuspended in 200/~1 of growth medium containing ~4C protein hydrolysate. After 12 h the cells were harvested and the 14C incorporation was measured and compared with uptake observed in the untreated cells. Results
The synthetic strategy adopted for the target compounds involved the prior synthesis of the appropriate phosphorochloridate [ROC(O)CHR'O][R"O]POC1, and its reaction with a suitable nucleoside analogue. This was based on the success of this strategy for simple, and halo-substituted, alkyl phosphate derivatives (McGuigan et al., 1990a,b). Thus, ethyl glycolate was prepared by the reaction of glycolic acid and ethanol in the presence of acid catalysis (Vogel, 1964), and ethyl phosphorodichloridate was prepared by the reaction of ethanol and phosphoryl chloride in the presence of triethylamine (Kosolapoff, 1950). Ethyl (ethyl glycolyl) phosphorochloridate (la) was prepared by the reaction of these reagents in ether at low temperature. The novel product gave a single resonance in the 3Xp NMR, at 6 + 3.3. This is slightly further upfield than the signal for the starting phosphorodichloridate, as would be expected (Mark et al., 1969). Similarly prepared by the reaction of ethyl glycolate with
207 O O I) jf Et--O--C--CH2--O--P--CI OR
0 HN~ /
o
o
la lb lc ld
R=Et R=nPr R=nHex R=C12Has
Me II
o~-..~
2aR=Et 2b R=nPr ,c
nH
x
0 Me 0
0
oH'~""N~J
o.I
3a R=nPr 3b R=n-C12H2s
°" ~_2 Na
0 O II It Me--O--C--CH-O--P--CI I I Me OR
4a R=Me 4b R=nPr
0 HN~ ' ~ , Me
o II
o ii
o~.Y
I
Me--O--C--CIH-- O-- IP-- 0--~ _OJ Me OR ~
5a R=Me 5b R=nPr
Na Scheme
1.
the appropriate alkyl phosphorodichloridates were the propyl and hexyl analogues (lb,c). These phosphorochloridates were used to phosphorylate nucleosides in pyridine at ambient temperature. In the first instance, phosphate derivatives (2a-c) of 3'-acetylthymidine were prepared. This not only served as a chemical model, but was also of some biological interest. In particular, we have suggested that suitable phosphorylation of nucleosides which are inactive against, for example, HIV (such as 3'-acetylthymidine) may yield active materials by a mechanism we term 'kinase by-pass' (McGuigan et al., 1990a,b).
208
Phosphorus-31 N M R analysis of the first homologue (2a) revealed two closelyspaced signals, at 6 1.6. The multiplicity corresponds to the presence of diastereoisomers resulting from the asymmetry of the phosphorus centre. The ratio of the signals (diastereoisomers) is not unity (2:1) indicating some preference for the formation (or isolation) of one stereoisomer from the racemic phosphorochloridate reacting with the optically pure nucleoside. In the case of (2b,c) the mixtures were separated into their pure diastereoisomers by chromatography (HPLC for 2b). The roughly equi-molar yields of the two isomers, in each case, indicated no such stereoisomeric preference as noted for (2a). The 13C N M R spectra of (2a-c) are particularly informative. In the case of (2a) the presence of the isomeric mixture leads to a complex spectrum. In particular, almost every signal is split into (at least) two closely spaced resonances, indicating the non-equivalence of the carbon atoms in the two isomers; including carbons ostensibly remote from the site of the mixed stereochemistry (the phosphate). The un-equal ratio of the two isomers in (2a) is useful, as it allows un-equivocal assignment of the signals for each individual isomer. The isomeric purities of (2b-c) by comparison, are evident from the relative simplicity of the 13C spectra. For each of (2a-c), in every case, where it can be resolved, phosphorus-coupling is noted for carbon atoms within 3 bonds of the phosphorus. The magnitude of the coupling varies, from 4 Hz (C = O) to 8 Hz (C4'). Similarly, 1H N M R data are complex for the mixed isomers of (2a), but are well resolved for the purified isomers of (2b~). The compounds were also characterised by H P L C (2a-c), FAB mass spectrometry (2b-c) and microanalysis (2c). It is interesting to note that, in both cases studied by mass spectrometry, the two isomers differ significantly in their fragmentation patterns. Propyl (ethyl glycolyl) phosphorochloridate (lb) was similarly allowed to react with the anti-HIV nucleoside analogue AZT (Mitsuya et al., 1985) to yield the target compound (3a). Again, the 3 p N M R spectrum clearly reveals the presence of two isomers in this sample. However, in this case the 1:1 ratio precluded assignment of each signal to each isomer. Compound (3a) was further characterised by HPLC, mass spectrometry, and microanalysis. At this stage it was considered to be of interest to prepare a derivative where the (simple) alkyl phosphate group was a long aliphatic chain. The rationale was that this would increase the lipophilicity of the pro-drug, and enhance membrane penetration (Gouyette et al., 1989). Thus, dodecyl (ethyl glycolyl) phosphorochloridate (ld) was prepared by the reaction of dodecyl phosphorodichloridate with ethyl glycolate. Compound (ld) was isolated in high yield, and characterised by 31p and 13C N M R and mass spectrometry. In the preparation of the previous AZT derivative (3a) a very extended reaction time (14 days) had been necessary in order to achieve complete reaction. It seemed likely that the reaction of (ld) to produce (3b) would be even slower, simply on steric grounds, and alternative reaction conditions were therefore employed. In particular, we have previously found (McGuigan et al., 1990c; Curley et al.,
209 1990) that THF/N-methylimidazole conditions of Van Boom and co-workers (Van Boom et al., 1975) lead to a rapid phosphorylation with even poorlyreactive phosphoramidates. Thus, reagent (ld) was allowed to react with AZT under these conditions. This gave a complete reaction (on TLC) after only 48 h, and (3b) was subsequently isolated in high yield, as a roughly 2:1 mixture of stereoisomers. It is interesting to note that this is also the ratio observed for the ethyl phosphate of acetylthymidine (2a), whereas the propyl (2b, 3a) and hexyl phosphates (2c) were produced in roughly equal isomeric ratios. It could be that there is a steric preference for one isomer when the alkyl (phosphate) substituent is substantially smaller, or larger, than the ethyl glycolyl moiety; which might have a steric requirement similar to a pentyl or hexyl chain. The structure and purity of (3b) was also confirmed by HPLC, mass spectrometry, and microanalysis. It was of interest to probe the effect on biological activity of branching on the a-carbon of the glycolyl moiety. This is analogous to comparing various amino acids in the phosphoramidate series (Devine et al., 1990). In particular, the synthesis of a-methyl glycolates (lactates) was pursued. Thus, S-(-)-methyl lactate was allowed to react with phosphoryl chloride in the presence of triethylamine to yield 31(methyllactyl) phosphorodichloridate in moderate yield. This revealed a single P N M R signal at 6 6.5; rather down-field of that noted above for simple alkyl phosphorodichloridates. In the 13C NMR, phosphoruscoupling was noted for carbon atoms within three bonds of the phosphorus, and 1H N M R and microanalysis data were also consistent with the structure and purity of the dichloridate. On reaction with methanol, this yielded methyl (methyl lactyl) phosphorochloridate (4a) in near-quantitative yield. On account of the chirality of the starting methyl lactate, compound (4a) was a mixture of diastereoisomers about the phosphorus centre, and gave two signals in the 31p NMR. The difference in chemical shift was quite large (ca 1 ppm), but no stereoselection was noted (1:1). Many of the 13C NMR signals were similarly split, and almost all further displayed phosphorus coupling. The structure and purity of phosphorochloridate (4a) was confirmed by mass spectrometry and microanalysis. To probe the effect of alkyl (phosphate) elongation in this series, propyl (methyl lactyl) phosphorochloridate (4b) was prepared, by the reaction of propyl phosphorodichloridate with methyl lactate. Again, 31p, 13C, and 1H NMR confirmed the presence of two stereoisomers, in a l:l ratio, and mass spectral data were also recorded. Phosphorochloridate (4a) was allowed to react with AZT in THF containing N-methylimidazole to give target compound (5a) in high yield. Two diastereoisomers were apparent from the 31p NMR, with a ratio of 3:2. It is interesting that the signals were significantly further upfield than previously noted for the analogous glycolyl compounds. Many of the 13C and ~H NMR signals were also split as a result of the isomeric nature of (5a). Similarly prepared was the propyl analogue (5b). Consistent with the suggestion above concerning the variation of the diastereomeric ratios, the ratio was now nearer
210 TABLE 1 Antiviral activity of derivatives reported Compound
EDso a
TDs0
2b 2c 3a 3b 5a 5b
> 100 > 100 0.5 3 0.6 10
NT NT > 100 75 > 100 > 100
ED50 is that concentration of drug in #M which reduces viral proliferation by 50%. The TDs0 is that concentration of drug which causes 50% toxicity to uninfected cells; for full details see methods section.
1:1. Other spectroscopic data confirmed the structure and purity of (5b). The acetylthymidine compounds (2b-c) and the AZT compounds (3a-b) and (5a-b) were tested against HIV-1 in vitro, using methods we have described (Kinchington et al., 1989), the results being given in Table 1.
Discussion
The acetylthymidine compounds (2b~) were entirely inactive at the highest concentration tested (100 ~M). Thus, the aim of activation via kinase by-pass is not successful in this instance. However, the AZT derivatives (3a-b) and (5a-b) were all inhibitors of viral proliferation. In each case, the activity of the compounds decreased on lengthening the alkyl phosphate chain (3a vs. 3b; 5a vs. 5b), or on substitution of an ethyl glycolate by a methyl lactate moiety (3a vs. 5b). In the latter case a direct comparison is possible, and suggests a difference in activity in the 2 series of approximately 20-fold. The reasons for these differences remain unclear. In the first case, the lengthening of the alkyl phosphate chain was designed to improve the lipophilicity, and hence membrane penetration, of the compounds. The inverse effect on biological activity suggests either that membrane penetration is not a limiting factor for the compounds, or that other detrimental effects overwhelm any such advantage. In the case of the glycolate/lactate comparison, the difference in activity is quite major. However, just such changes in activity have been noted for phosphoramidate derivatives, where the amino acid side-chain (analogous to the site of alteration here) is modified only slightly (McGuigan et al., 1990c; McGuigan et al., 1991). In order to further probe the origins of this difference in activity, compounds (3a) and (5b) were incubated in growth medium at 37°C, and at 5°C, with controls in DMSO/water. At 37°C, both nucleotides decomposed entirely over a period of one week when dissolved in growth medium, with only 6% decomposition in DMSO/water under similar conditions. In growth medium at lower temperature, the decomposition was somewhat reduced (25% unchanged after 1 week). In the cases where decomposition was complete, HPLC revealed
211
the generation of AZT, but only in trace amounts (ca 0.05%). Thus, unless the release of AZT were greatly facilitated in vitro, it would seem likely that nucleoside release is not the primary mode of action of these compounds. On the other hand, major quantities of several very polar compounds were generated from (3a) and (5b), particularly in growth medium at 37°C. The precise structures of these hydrolysis products were not determined, but their mobility on TLC and HPLC suggests that they are free nucleotides or polar derivatives thereof. No significant differences could be noted in the hydrolysis of the two derivatives (3a) and (5b), either in a qualitative or quantitative sense. Thus no explanation of the 20-fold difference in their activities was apparent from the hydrolysis study; suggesting the involvement of some in vitro parameter in this distinction. In conclusion, we have demonstrated that phosphate triester derivatives of AZT are moderate to potent inhibitors of HIV when one of the alkyl chains is ester-containing; in particular is glycolate or lactate. Ethyl glycolate appears to be more efficacious in this regard than methyl lactate. Lengthening of the remaining alkyl phosphate group appears to lead to a reduction in biological activity. The origins of the differences in activity in the glycolate/lactate series do not appear to correspond to simple hydrolytic lability, and the mechanisms by which the compounds act remain unclear. However, the general level of activity of the ester-substituted compounds (3a-b) and (5a-b) is consistent with a mechanism of action involving intracellular delivery of the free nucleotide, or a close derivative thereof.
Acknowledgements CN thanks the SERC for a studentship. CMc thanks Mr. S. Corker (University College London) and Mr. S.J. Turner (Southampton) for HPLC analyses. DK thanks the MRC and Roche products U.K. Ltd for financial support.
References Bundgaard, H. (1985) Design of prodrugs. Elsevier, Amsterdam. Chawla, R.R., Freed, J.J. and Hampton, A. (1984) Bis(m-nitrophenyl) and bis(p-nitrophenyl) esters and the phosphorodiamidate of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in mouse cells in culture. J. Med. Chem. 27, 1733-1736. Curley, D., McGuigan, C., Devine, K.G., O'Connor, T.J., Jeffries, D.J. and Kinchington, D. (1990) Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity. Antiviral Res. 14, 345-356. Devine, K.G., McGuigan, C., O'Connor, T.J., Nicholls, S.R. and Kinchington, D. (1990) Novel phosphate derivatives of zidovudine as anti-HIV compounds. AIDS 4, 371-372. Farquhar, D. and Smith, R. (1985) Synthesis and biological evaluation of 9-[5'-(2-oxo-l,3,2oxazaphosphorinan-2-yl)-/~-D-arabinosyl] adenine and 9-[5'-(2-oxo-l,3,2-dioxaphosphorinan-2yl)-fl-D-arabinosyl] adenine: potential neutral pre-cursors of 9-[/~-D-arabinosyl] adenine 5'monophosphate. J. Med. Chem. 28, 1358-1361.
212 Gouyette, C., Neumann, J.M., Fauve, R. and Huynh-Dinh, T. (1989) 6- and l-substituted mannosyl phosphotriesters as lipophilic macrophage-targeted carriers of antiviral nucleosides. Tetrahedron Lett. 30, 6019 6022. Hunston, R.N., Jones, A.S., McGuigan, C., Walker, R.T., Balzarini, J. and DeClercq, E. (1984) Synthesis and biological properties of some cyclic phosphotriesters derived from 2'-deoxy-5fluorouridine. J, Med. Chem. 27, 440-444. Jones, B.C.N.M., McGuigan, C. and Riley, P.A. (1989) Synthesis and biological evaluation of some phosphate triester derivatives of the anti-cancer drug araC. Nucleic Acids Res. 17, 7195-7201. Kinchington, D., Galpin, S.A., O'Connor, T.J., Jeffries, D.J. and Williamson, J.D. (1989) Comparison of antigen capture assays against HIV-1 in evaluating anti-viral compounds. AIDS 3, 101-104. Kosolapoff, G.M. (1950) Organophosphorus compounds. Wiley, New York. Mark, V., Dungan, C.H., Crutchfield, M.M. and Van Wazer, J.R. (1969) Compilation of 3~p NMR data. In: M. Grayson and E.J. Griffith (Eds) Topics in Phosphorus Chemistry, vol. 5, Wiley, New York. McGuigan, C., Tollerfield, S.M. and Riley, P.A. (1989) Synthesis and biological evaluation of some phosphate triester derivatives of the anti-viral drug araA. Nucleic Acids Res. 17, 6065-6075. McGuigan, C., Nicholls, S.R., O'Connor, T.J. and Kinchington, D. (1990a) Synthesis of some novel dialkyl phosphate derivatives of 3'-modified nucleosides as potential anti-AIDS drugs. Antiviral Chem. Chemother. 1, 25 33. McGuigan, C., O'Connor, T.J., Nicholls, S.R., Nickson, C. and Kinchington, D. (1990b) Synthesis and anti-HIV activity of some novel substituted dialkyl phosphate derivatives of AZT and ddCyd. Antiviral Chem. Chemother. 1,355 360. McGuigan, C., Devine, K.G., O'Connor, T.J., Galpin, S.A., Jeffries, D.J. and Kinchington, D. (1990c) Synthesis and evaluation of some novel phosphoramidate derivatives of 3'-azido-3'deoxythymidine (AZT) as anti-HIV compounds. Antiviral Chem. Chemother. 1, 107-113. McGuigan, C., Devine, K.G., O'Connor, T.J. and Kinchington, D. (1991) Synthesis and anti-HIV activity of some haloalkyl phosphoramidate derivatives of 3'-azido-3'-deoxythymidine (AZT): potent activity of the trichloroethyl methoxyalaninyl compound. Antiviral Res. 15, 255 263. Mitsuya, H., Weinhold, K.J., Furman, P.A., St. Clair, M.H., Nusinoff-Lehrman, S., Gallo, R.C., Bolognesi, D., Barry, D.W. and Broder, S. (1985) 3'-Azido-3'-deoxythymidine(BWA509U): An agent that inhibits the infectivity and cytopathic effect of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci. USA 82, 7096-7100. Van Boom, J.H., Burgers, P.M.J., Crea, R., Luyten, W.C.M.M. and Reese, C.B. (1975) Phosphorylation of nucleoside derivatives with aryl phosphoramidochloridates. Tetrahedron 31, 2953-2959. Vogel, A.I. (1964) Practical organic chemistry. 3rd Edn, Longmans, London.
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© 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-3542/92/$05.00 AVR 00530
Synthesis and anti-HIV activity of some novel lactyl and glycolyl phosphate derivatives Christopher McGuigan l, Caleb Nickson 2, Timothy J. O'Connor 3 and Derek Kinchington 3 IDepartment of Chemistry, University of Southampton, Highfield, Southampton, 2Department of Chemistry, University College London, London and 3Department of Virology, Medical College of St. Bartholomew's Hospital, Bartholomew Close, London, U.K. (Received 11 July 1991; accepted 7 August 1991)
Summary Novel phosphate triester derivatives of 3'-acetylthymidine, and of the antiHIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. These materials are designed to act as membrane-soluble pro-drugs of the bio-active free nucleotides. In particular, novel glycolate and lactate phosphate derivatives have been prepared. In vitro evaluation revealed the AZT compounds to have a pronounced and selective antiviral effect, the magnitude of which varied considerably with the nature of the phosphate blocking group. HIV; AZT; Nucleotide; Pro-drug; Lactate; Glycolate
Introduction Nucleoside analogues are firmly established as useful agents to combat viral infection and in the chemotherapy of cancer. However, they have a number of limitations, which might in part be overcome by the use of masked phosphate derivatives, if it is possible to release the bio-active free nucleotides intracellularly (Hunston et al., 1984; Chawla et al., 1984; Farquhar and Smith, 1985). We have applied this idea to the anti-herpetic agent araA (McGuigan et al., 1989) and the anti-cancer drug araC (Jones et al., 1989). Correspondence to." C. McGuigan, Dept. of Chemistry, University of Southampton, Highfield, Southampton, SO9 5NH, U.K.
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More recently, we have found that simple dialkyl phosphate derivatives of AZT, and other nucleoside analogues, are inactive as anti-HIV agents (McGuigan et al., 1990a), whereas trihaloethyl phosphates are active (McGuigan et al., 1990b). It was of interest to investigate whether other types of phosphate blocking groups were effective in this capacity. In this paper we disclose the observation that ester-containing phosphate protecting groups are particularly efficacious. We were prompted to investigate ester-containing groups by several factors. Firstly, we have noted that C-protected, N-linked amino acids are suitable blocking groups, leading to a significant anti-viral effect in many cases (Devine et al., 1990; McGuigan et al., 1990c). In the case of glycine compounds, the esters we will describe below are very closely related to these phosphoramidate analogues, with the link to the phosphate simply being altered from P-NH to P-O. Secondly, intracellular cleavage of the co-esters by carboxyl esterase would yield co-carboxylates. These may be trapped within the cell on the grounds of their increased polarity, and their phosphate hydrolysis may well be enhanced by intramolecular participation of the free carboxylate function. Lastly, it is noteworthy that ester containing groups are frequently used in pro-drug design, particularly as alcohol blocking groups (Bungdaard, 1985).
Materials and Methods
Commercially available Merck kieselgel F254 plates were used for the TLC and the components were visualised by UV light. Column chromatography was carried out using Woelm silica (32-63 mm) as the stationary phase. The ratio of silica-compound varied between 50:1 and 100:1 (w/w). EI MS was carried out by Dr. M. Mruzek on a VG 7070H mass spectrometer fitted with a Finnigan Incos II data system. FAB MS was carried out by the University of London mass spectrometry service on a VG Zab 1F spectrometer. Analytical HPLC was carried out on an ACS quaternary system, using a Spherisorb CN5~M column and an eluant of 5% water in MeCN, with a flow rate of 2 ml/min and detection by UV at 265 nm. Preparative HPLC was conducted on a Waters system using a Partisil 5 silica column and a mobile phase of 90% ethyl acetate/ 10% petroleum spirit, with a flow rate of 2 ml/min. Analytical data were obtained for solid products, courtesy of the team of Mr. A. Stones (University College London); oils and gums did not analyse within accepted limits, but were pure by spectroscopy, mass spectrometry, and HPLC. Phosphorus-31 N M R spectra of phosphorylating reagents were recorded on a Varian XL-200 spectrometer operating at 82 MHz. Phosphorus-31 NMR spectra of phosphate triesters were recorded on a VXR-400 spectrometer operating at 164 MHz and are reported in units of 6 relative to 85% phosphoric acid as external standard, positive shifts are downfield. Carbon-13 NMR spectra were recorded on a Varian XL-200 spectrometer operating at 50 MHz for all compounds except nucleotides, which were recorded on a Varian VXR-400 spectrometer operating
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at 100 MHz and are reported in units of ~ relative to CDC13 at 77000 ppm. Both phosphorus-31 and carbon-13 N M R spectra were proton noise decoupled and all signals were singlets unless otherwise stated. H-1 N M R spectra were recorded on a Varian XL-200 spectrometer operating at 200 MHz for all nonnucleoside containing compounds which were analysed with a Varian VXR-400 spectrometer operating at 400 MHz and are reported in units of 6 relative to internal CHC13 at 7.240 ppm unless otherwise stated. All N M R spectra were recorded in CDC13. All experiments involving water-sensitive reagents were carried out under scrupulously dry conditions. Where needed, anhydrous solvents and reagents were obtained in the following ways: pyridine was heated under reflux over calcium hydride for several hours, distilled and stored over activated molecular sieves. N-methylimidazole was heated to 100°C over Call2 for 4 h, then distilled under reduced pressure. Tetrahydrofuran was refluxed over potassium for several hours, distilled and stored over activated molecular sieves. Phosphoryl chloride was distilled prior to use.
Ethyl (ethyl glycolyl) phosphorochloridate ( la) Ethyl glycolate (2.91 g, 2.91 ml, 0.03 mol) and triethylamine (2.64 g, 3.64 ml, 0.03 mol), in diethyl ether (100 ml) were added dropwise over 1 h to a vigorously stirred solution of ethyl phosphorodichloridate (4.68 g, 3.41 ml, 0.03 mol) in diethyl ether at -60°C. After stirring for 4 h at this temperature the reaction mixture was allowed to warm to room temperature and left stirring overnight. Filtration and concentration under reduced pressure, followed by hexane (ca 50 ml) extraction afforded an oil (4.75 g, 79%). 6p 3.343.
Propyl (ethyl glycolyl) phosphorochloridate (lb) This was prepared by a method analogous to that used for (la) above. Thus, from 2.91 g (0.03 mol) of ethyl glycolate was isolated 4.03 g (62%) of (lb). fie 3.163.
Hexyl (ethyl glycolyl) phosphorochloridate (lc) This was prepared by a method analogous to that used for (la) above. Thus, from 0.24 g (2 mmol) of ethyl glycolate was isolated 0.6 g (92%) of (lc). fie 3.841; 6H 4.725 (2H, d, C(O)CH20, J=4.1 Hz), 4.27 (4H, m, CH2OC(O), CH2CH_H_20), 1.754 (2H, quintet CH2CH2CH20, J=7.25 Hz), 1.32 (9H, m, CHsCH2OC , CH3(CH___2)2CH2CH2CH20), 0.902 (3H, t, C H3CH2CH2, J = 7.14 Hz); found C 42.30%, H 7.05, P 10.68, C1 11.88; CloH2oOsPCI requires C 41.89%, H 7.03, P 10.80, C1 12.37.
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Dodecyl (ethyl glycolyl) phosphorochloridate (ld) This was prepared by a method analogous to that used for (la) above, except that an initial reaction temperature of -30°C was used. Thus, from 2.0 g (0.02 mol) of ethyl glycolate was isolated 7.1 g (96%) of (ld). 6c 166.393 (d, C(O), J = 8.1 Hz), 70.575 (d, CHzCH20, J=7.7 Hz), 63.899 (d, C(O)CH2OP, J = 5.9 Hz), 61.841 (CH2OC), 31.806 (_CHzCH2CH20), 29.512-22.586 ([_C__H2]s,m), 22.586 (CH3CH2CH2), 14.010 (_CH3CH20), 13.920(CH3R); 6p 3.577; m/e (EI) 373 (MH +, ~C1, 1.47%), 371.1754 (MH +, C16H33OsPC1 requires 371.1754, 5.00), 335 (M + -C1, 0.92), 271 (M + -CH3[CH2]6, 8.14), 205 (MH2 + - C H 3 [CH2]ll, 37C1, 35.43), 203 (MH2 + - CH3[CH2]ll, 100), 111 ([CHz]vCH +, 4.33), 83 ([CHz]sCH +, 10.14), 69 ([CHz]4CH +, 18.40), 55 ([CHz]3CH +, 33.28), 41 ([CHz]2CH +, 47.13).
3'-O-Acetylthymidine-5'-ethyl (ethyl glycolyl) phosphate (2a) To a stirred solution of 3'-O-acetylthymidine (0.61 g, 0.002 mol), in pyridine (10 ml) was added phosphorochloridate (la) (1.38 g, 1.27 ml, 0.06 mol). After leaving for 96 h., the reaction mixture was concentrated to dryness and triturated with toluene (ca 3 x 10 ml). Purification was achieved by flash column, chromatography on silica (ca 100 g) eluted with 5% methanol in chloroform. Pooling and evaporation of appropriate fractions afforded a colourless gum (0.22 g, 24%). 6c 170.389 (CH3C(O), A), 170.368 (CH3C(O), B), 167.862 (d, C(O)CH2, J=4.3 Hz, B), 167.608 (d, C(O)CH2, J=4.7 Hz, A), 163.927 (C-2, A), 163.926 (C-2, B) 150.666 (C-4, A), 150.650 (C-4, B), 135.026 (C-6, A), 134.926 (C-6, B), 111.612 (C-5, A), 111.562 (C-5, B), 84.317 (C-I', B), 84.242 (C-I', A), 82.637 (d, C-4', J=8.0 Hz, B), 82.554 (d, C-4', J=8.2 Hz, A), 74.484 (C-3', B), 74.464 (C-3', A), 67.257 (d, C-5', J = 5.2 Hz, B), 67.190 (d, C5', J=5.7 Hz, A), 64.758 (d, C(O)C__H2OP, J=5.3 Hz, B), 64.699 (d, C(O)CH2OP, J=6.0 Hz, A), 63.850 (d, CH3CH2OP, J--5.3 Hz, B), 63.660 (d, CH3CHzOP, J = 7.0 Hz, A), 61.648 (CH3CH2OC, A), 61.590 (CH3CH2OC, B), 36.993 (C-2', B), 36.931 (C-2', A), 20.776 (CH3C(O)), 15.891 (m, CH3CHzOP), 13.929 (CH3CH2OC), 12.402(5-Me); 6p 1.723, 1.548 (A:B; 2:1); 6/-/9.897 (1H, s, NH), 7.476, 7.458 (1H, d, H-6, J-- 1.22, 1.17 Hz), 6.36 (1H, m, H-I'), 5.27 (1H, m, H-3'), 4.57 (2H, m, C(O)CH20), 4.36 (2H, m, H-5'), 4.2294.005 (5H, m, CH3CH2OC, H-4', CH3CH_zOP), 2.35 (1H, m, H-2'), 2.16 (1H, m, H-2'), 2.052 (1H, s, CH3C(O)), 1.869, 1.872 (3H, d, base CH3, J = 1.09, 1.13 Hz), 1.37 (3H, m, CH3CH2OP), 1.218 (3H, t, CH__3CH2OC, J=7.68 Hz).
3'-O-A cetylthymidine-5'-(propyl ethyl glycolyl) phosphate (2b) This was prepared by a method entirely analogous to that used for (2a) above, except that the reaction was stirred for 70 h, and the chromatographic column was eluted much more slowly (0.5% methanol in chloroform), allowing a partial separation of the diastereoisomers; the pure diastereoisomers being
201 isolated as oils by preparative HPLC. Thus, from 0.54 g of 3'-0acetylthymidine was isolated 0.31 g (32%) of (2b).
Fast isomer (0.16 g, 16%). 6c 170.389 (CH3C(O)), 167.736 (d, C(O)CH2, J=4.8 Hz), 163.554 (C-2), 150.450 (C-4), 135.154 (C-6), 111.754 (C-5), 84.394 (C-I'), 82.794 (C-4', J=8.4 Hz), 74.628 (C-3'), 70.091 (d, CH2CH20, J=6.1 Hz), 67.321 (d, C-5', J = 5.7 Hz), 63.666 (d, C(O)CHzOP, J=5.0 Hz), 61.734 (CH3CH2OC), 37.089 (C-2'), 23.572 (d, CH2CH2OP, J=7.1 Hz), 20.896 (CH3C(O)), 14.073 (_CH3CH2OC), 12.329 (base CH3), 9.868 (_QH3CHzCH20); 6,0 0.776; 6/¢ 8.836 (1H, s, NH), 7.483 (1H, d, H-6, J = 1.26 Hz), 6.358 (1H, q, H-I'), 5.257 (1H, d, H-3', J = 5.98 Hz), 4.56 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.170 (2H, q, CH3CH__2OC, J=6.56 Hz), 4.12 (1H, m, H-4'), 4.05 (2H, m, CHzCH__20), 2.34 (1H, m, H-2'), 2.22 (1H, m, H-2'), 2.091 (3H, s, CH3C(O)), 1.877 (3H, d, base CH3, J = 1.01 Hz), 1.674 (2H, sextet, CH3CHHzCHzO, J = 6.77 Hz), 1.218 (3H, t, CH3CHzOC, J=7.32 Hz), 0.901 (3H, t, CH3CH2CH2, J = 7.36 Hz); m/e (FAB) 493 (MH +, 0.05%), 492 (M +, 0.01), 102 (C4H603 +,
lOO). Slow isomer (0.15 g, 16%). 6c 170.409 (CH3__C(O)), 167.719 (d, C(O)CH20, J=4.6 Hz), 163.715 (C-2), 150.378 (C-4), 135.249 (C-6), 111.687 (C-5), 84.561 (C-I'), 82.833 (d, C-4', J = 8.3 Hz), 74.589 (C-3'), 70.187 (d, CH2CH20, J = 5.9 Hz), 67.374 (d, C-5' J=6.0 Hz), 63.753 (d, C(O)CH2, J=5.4 Hz), 61.753 (_C_H2OC), 37.198 (C-2'), 23.540 (d, CH2CH20, J = 6.8 Hz), 20.896 (_CH3C(O)), 14.076 (C_H3CH20), 12.294 (base CH3), 9.866 (_C_H3CH2CH2); 6e 0.362; 5z4 8.879 (1H, s, NH), 7.463 (1H, d, H-6, J = 1.22 Hz), 6.357 (1H, q, H-I', J = 6.78 Hz), 5.282 (1H, d, H-3', JH-4'= 5.02 Hz), 4.56 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.183 (2H, q, CH3CH_2OC, J = 6.71 Hz), 4.12 (1H, m, H-4'), 4.05 (2H, m, CH2CH2OP), 2.34 (1H, m, H-2'), 2.22 (1H, m, H-2'), 2.091 (3H, s, CH3C(O)), 1.878 (3H, d, base CH3, J = 0.98 Hz), 1.668 (2H, sextet, CH3CH_zCH20, J = 6.69 Hz), 1.224 (3H, t, CH3CH2OC, J = 7.30 Hz), 0.895 (3H, t, CH3CH2OP, J = 7.36 Hz); m/e (FAB) 493 (MH +, 0.01%), 492 (M +, 0.01), 73 (C2H5OCO +, 100). 3'-O-Acetylthymidine-5'-hexyl (ethyl glycolyl) phosphate (2c) This was prepared by a method entirely analogous to that used for (2b) above, except that the chromatographic column was eluted with 0.55% methanol in chloroform, which gave a near-complete separation of the diastereoisomers. HPLC indicated the 'fast' isomer to be pure, whilst the 'slow' isomer contained 3% of the other diastereomer. Thus, from 0.67 g of 3'-0acetylthymidine was isolated 0.66 g (58%) of (2c) as a solid.
Fast Isomer (0.36 g, 32%). 6c 170.470 (CH3C(O)), 167.69 (d, C(O)CH2, J=4.9 Hz), 163.735 (C-2), 150.564 (C-4), 135.041 (C-6), 111.778 (C-5), 84.495 (C-I'), 82.854 (d, C-4', J = 8.2 Hz), 74.629 (C-3'), 68.882 (d, CHzCH2OP, J = 6.1 Hz), 67.340 (d, C-5', J = 5.2 Hz), 63.702 (C(O)CH2OP), 61.731 (CH3CH2OC),
202
37.175 (C-2'), 30.173 (d, CH2CH2OP, J=6.1 Hz), 29.688 (_C_HzCH2CH20), 25.605 (CH3CHzCH2), 22.464 (CH3C_H2CH2), 20.887 (CH3C(O)), 14.099 (C_H3CH2OC), 13.928 (_C_H3CHzCH2), 12.232 (base CH3); fie 1.782; 6,4 9.009 (1H, broad s, NH), 7.502 (1H, d, H-6, J = 1.30 Hz), 6.42 (1H, m, H-I'), 5.31 (1H, m, H-3'), 4.62 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.23 (2H, m, CH3CH__2OC), 4.18 (1H, m, H-4'), 4.15 (2H, m, CHzCH2OP), 2.40 (1H, m, H2'), 2.38 (1H, m, H-2'), 2.07 (3H, s, CH3C(O)), 1.932 (3H, s, base CH3), 1.69 (2H, q, CH2CHzCH20, J=6.65 Hz), 1.25 (I1H, m, CH3CH2OC, (CH2)4), 0.928 (3H, t, CH__3CHzCH2, J=7.67 Hz); m/e (FAB) 536 (MH2 +, 0.5%), 535 (MH +, 6), 433 (3), 267 (acetylthymidine. H + - H 2 0 , 5), 147 (C6H1202 p+, 100), 81 (C5H50 +, 45).
Slow isomer (0.30 g, 26%). 6c 170.518 (CH3C(O)), 167.708 (d, C(O)CH2, J=4.9 Hz), 163.835 (C-2), 150.624 (C-4), 135.081 (C-6), 111.748 (C-5), 84.361 (C-I'), 82.757 (d, C-4', J = 8.2 Hz), 74.624 (C-3'), 68.240 (d, CH2CHzOP , J = 6.1 Hz), 67.331 (d, C-5', J = 5.2 Hz), 63.733 (C(O)CHzOP), 61.726 (CH3CH2OC), 37.124 (C-2'), 30.173 (d, CH2CHzOP J=6.1 Hz), 29.633 (_C_HzCH2CH20), 25.835 (CH3CH2CH2), 22.962 (CH3CHzCH2), 20.826 (_C_H3C(O)), 14.048 (_C_H3CH2OC), 13.238 QC_H3CH2CH2), 12.312 (base CH3); fie 1.625; 6/-/9.282 (1H, broad s, NH), 7.535 (1H, d, H-6, J = 1.30 Hz), 6.32 (1H, m, H-I'), 5.323 (1H, m, H-3'), 4.61 (2H, m, C(O)CH20), 4.34 (2H, m, H-5'), 4.23 (2H, m, CH3CH2OC), 4.17 (1H, m, H-4'), 4.15 (2H, m, CH2CHzOP), 2.38 (1H, m, H2'), 2.26 (1H, m, H-2'), 2.07 (3H, s, CH3C(O)), 1.902 (3H, d, base CH3, J = 1.16 Hz), 1.66 (2H, m, CH2CH2CH20) 1.24 (11H, m, CH3CH2OC, (CH2)4), 0.902 (3H, t, CH3CHzCH2, J=7.67 Hz); m/e (FAB) 536 (MHz +, 1.0%), 535 (MH + 8), 433 (9), 267 (acetylthymidine. H + --H20, 6), 149 (C6H1402 p+, 100), 8; (C5H50 +, 78); found C 49.70%, H 6.91, N 4.79, P 5.01; CzzH35NzOllP requires C 49.44, H 6.60, N 5.24, P 5.79. 3'-Azido-3'-deoxythymidine-5'-propyl (ethyl glycolyl) phosphate (3a) Phosphorochloridate (lb) (1.10 g, 0.004 mol) was added to AZT (0.2 g, 0.75 mmol) in pyridine (10 ml) with stirring. After leaving for 14 days, water (ca 2 ml) was added and the reaction mixture concentrated to dryness and triturated with toluene (3 × 20 ml), Purification by flash column chromatography (silica ca 60 g) eluted with 3% methanol in chloroform gave a solid (0.13 g, 37%); 6c 167.785 (d, C(O)CH2 J = 4.4 Hz), 167.716 (d, C(O)CH2, J = 4.8 Hz), 163.913 (C2), 150.392 (C-4), 135.191 (C-6), 135.073 (C-6), 111.395 (C-5) 111.352 (C-5), 84.516 (C-I'), 84.469 (C-I'), 82.144 (d, C-4', J=8.3 Hz), 70.128 (d, CHzCH20, J=6.6 Hz), 70.062 (d, CHzCH20, J=6.5 Hz), 66.573 (d, C-5', J=5.8 Hz), 66.440 (d, C-5', J=5.3 Hz), 63.717 (d, C(O)CH2, J=4.3 Hz), 63.639 (d, C(O)CH2 J = 5.1 Hz), 61.683 (_C_H2OC), 60.110 (C-3'), 60.051 (C-3'), 37.395 (C2'), 37.326 (C-2'), 23.415 (d, CH3CH2CH2, J=7.0 Hz), 13.971 (CH3CH20), 12.290 (base CH3), 12.263 (base CH3), 9.767 (_C_H3CH2CH2); 6p 0.766, 0.362 (A:B, 1:1); 6/~ 9.732 (1H, s, NH), 7.394, 7.379 (1H, d, H-6, J = 1.26, 1.19 Hz),
203 6.21 (1H, m, H-I'), 4.58 (2H, m, C(O)CH2) 4.39 (1H, m, H-3'), 4.34 (2H, m, H5'), 4.18 (2H, m, CHH_2OP),4.04 (2H, m, CH2OC), 3.98 (1H, m, H-4'), 2.43 (2H, m, H-2'), 1.863, 1.855 (3H, d, base CH3, J--1.07, 1.07 Hz), 1.65 (2H, m, CHaCH__2CH2) , 1.226 (3H, t, C H 3 C H 2 0 , J=7.17 Hz), 0.899 (3H, t, C H a C H E C H 2 , J=7.36 Hz); m/e (EI) 476 (MH + 5.91%), 373 (1.01), 350 (MH +-thymine, 1.85), 329 (AZTPOeH +, 0.98), 307 (MH2 +-thymine - Nail, 36.90), 250 (AZT.H + - - H 2 0 , 17.19), 209 (M+-AZT, 4.36), 185 (18.41) 167 (PrO[EtO]PO2, 14.56), 139 (PrOPOaH, 54.71), 127 (thymine H +, 23.29), 126 (thymine +, 30.36), 123 (PrOPO2H, 18.71), 81 (C5H50 +, 100); found C 42.80%, H 5.39, N 14.25, P 6.45; C17H2609N5 P requires C 42.95, H 5.51, N 14.73, P 6.52.
3'-Azido-3'-deoxythymidine-5'-dodecyl (ethyl glycolyl) phosphate (3b) Phosphorochloridate (ld) (1.66 g, 0.004 mol), was added to AZT (0.2 g, 0.007 mol) and N-methylimidazole (0.49 g, 0.48 ml, 0.006 mol) in tetrahydrofuran (2.5 ml) at room temperature. After five min a further 10 ml of tetrahydrofuran was added to the solidified mixture. After leaving for two days the reaction mixture was concentrated to dryness under reduced pressure, dissolved in chloroform (35 ml) and saturated sodium bicarbonate solution (15 ml) was added. The emulsion was concentrated to dryness and dissolved in chloroform (ca 4 ml). This was added portionwise to a stirred solution of petroleum ether (b.p. 30-40°C) (ca 500 ml) and the mixture refrigerated overnight at - 20°C. Further purification of the product was achieved by flash column chromatography (silica ca 60 g) eluted with 4% methanol in chloroform, pooling and evaporating the appropriate fractions. Final purification was achieved by flash chromatography (silica ca 60 g) eluted with chloroform. Pooling and evaporating the appropriate fractions gave the product as a solid (0.36 g, 79%). 6c 167.793 (d, C(O)CH2, A, J=4.3 Hz), 167.717 (d, C(O)CH2, B, J=4.6 Hz), 163.855 (C-2, A), 163.838 (C-2, B), 150.391 (C-4), 135.155 (C-6, A), 135.033 (C-6, B), 111.425 (C-5, A), 111.382 (C5, B), 84.509 (C-I', A), 84.453 (C-I', B), 82.166 (d, C-4', J=8.1 Hz), 68.755 (d, C H z C H z C H 2 0 , A, J=6.1 Hz), 68.678 (d, CHzCH2CH20, B, J=5.5 Hz), 66.564 (d, C-5', A, J=5.9 Hz), 66.444 (d, C-5', B, J = 5.3 Hz), 63.723 (d, C(O)CH2, A, J = 5.2 Hz), 63.643 (d, C(O)CHz, B, J = 5.1 Hz), 61.682 (_C_H2OC), 60.124 (C-3', A), 60.066 (C-3', B), 37.435 (C-2', A), 37.356 (C-2', B), 31.771 (_CH2CHzCH20), 30.059 (d, CH2CH2CH20, J=6.9 Hz), 29.486-25.220 (m, (_~_H2)8), 22.555 (CH3CHzCH2), 13.999 (C_H3CH20), 13.985 (_C_H3R), 12.320 (5Me, A), 12.293 (5-Me, B); 6p 0.510, 0.137 (A:B, 7:3); 6/_/9.7 (1H, s, NH), 7.428, 7.411 (d, 1H, H-6, A, B, J=1.22, 1.19 Hz), 6.253, 6.250 (1H, t, H-I', A, B, J=7.31, 6.95 Hz), 4.60 (2H, m, C ( O ) C H 2 ) , 4.42 (1H, m, H-3'), 4.36 (2H, m, H5'), 4.22 (2H, m, CH2OP), 4.102 (2H, q, CH2OC, J=6.9 Hz), 4.01 (1H, m, H4'), 2.38 (1H, m, H-2'), 2.31 (1H, m, H-2'), 1.895, 1.888 (3H, d, 5-Me, B, A, J = 1.01, 0.95 Hz), 1.66 (2H, m, CHzCH20), 1.26 (3H, m, CH3CH20), 1.215 (18H, m, ( C H 2 ) 9 ) , 0.838 (3H, t, CH3CHzCH2, J = 6.68 Hz); m/e (El) 433 (MH +
204
-C12H25, 26.79%), 250 (AZT.H + - H 2 0 , 18.25), 186 (C12H25OH +, 6.87), 185
(C12H25O+, 78.68), 167 (C4HyO3PO2H +, 13.34), 127 (30.11), 126 (thymine.H +, 49.14), 113 (127 -CH2, 18.63), 99 (113 -CH2, 22.85), 85 (99CH2, 11.73), 81 (C5H50 +, 100), 71 (85 -CH2, 27.54), 57 (71 -CH2, 64.23), 43 (57 --CH2, 85.41), 29 (C2H5+, 80.26); m/e (FAB) 603 (MH2 +, 2%), 602 (MH +, 8), 434 (MH2 + --C12H25, 0.5), 433 (MH + --C12H25, 1), 353 (M + + O H - A Z T , 1), 250 (AZT.H + -H20, 8), 185 (C12H250+, 11), 81 (C5H50 +, 100); found C 51.49%, H 7.41, N 11.54, P 5.19; C26H44N509P requires C 51.91, H 7.37, N 11.64, P 5.15.
Methyl lactyl phosphorodichloridate (S)-( - )-Methyl lactate (8.72 g, 8.00 ml, 0.08 mol) and triethylamine (8.46 g, 11.6 ml, 0.08 mol) in diethyl ether (200 ml) were added to a vigorously stirred solution of phosphoryl chloride (12.9 g, 7.83 ml, 0.08 mol) in diethyl ether (200 ml) at -78°C. The reaction was kept at this temperature for a further 12 h with stirring. After warming to room temperature, filtration and concentration under reduced pressure gave an oil that was distilled (67-70°C/0.05 mm Hg) to afford the product as a colourless oil (12.57 g, 68%). 6c 168.405 (d, C(O), J=6.0 Hz), 75.309 (d, CH, J=8.8 Hz), 52.747 (CH30), 18.543 (d, CH3CH, J=6.7 Hz); 6p 6.458; 61-/5.12 (1H, m, CH), 3.759 (3H, s, CH30), 1.629 (3H, d x d , CH3C, Jp= 1.05 Hz, JcH=6.8 Hz); found C 21.66%, H 3.19, P 15.01; C4H704PC12 requires C 21.74, H 3.19, P 14.02.
Methyl (methyl laetyl) phosphorochloridate (4a) This was prepared by a method entirely analogous to that used for methyl lactyl phosphorodichloridate above, except that a reaction temperature of -78°C was used for 12 h, followed by 14 h at -40°C. Thus, from 2.2 g of methyl lactyl phosphorodichloridate was produced 2.03 g (94%) of crude (4a). 6c 169.325 (d, C(O), J=5.4 Hz), 169.095 (d, C(O), J=6.2 Hz), 73.179 (d, CH, J=2.3 Hz), 73.051 (d, CH, J=2.0 Hz), 55.601 (d, CH3OP, J--7.1 Hz), 52.366 (CH3OC), 18.611 (d, CH3CH, J=5.4 Hz), 18.392 (d, CH3CH, J=6.2 Hz); 6e 4.001, 3.217 (A:B, 1:1); m/e (EI) 219 (MH +, 37C1, 0.07%), 217.005 (MH +, CsHIIOsPC1 requires 217.003, 0.39), 185 (MH + -CH3OH, 3.34), 181 (M + -C1, 13.72), 157 (MH + -C2H402, 100), 133 (CH3OPO2HzC1 + , 37 C1, 21.03), 131 (CH3OPO2HzC1 +, 67.74), 115 (CH3OPOCI +, 37C1, 8.60), 113 (CH3 OPOC1 +, 25.73); found C 26.86%, H 4.56, P 14.09; CsH10POsC1 requires C 27.73, H 4.65, P 14.30.
Propyl (methyl lactyl) phosphorochloridate (4b) This was prepared by a method entirely analogous to that used for (4a) above, except that a reaction temperature of -60°C was used for 4 h, followed by 14 h at ambient temperature. Thus, from 4.369 of (S)-( - )-methyl lactate
205 was produced 6.36 g (62%) of (4b) (b.p. 128°C/0.01 mmHg). 6c 169.590 (d, C(O), J=5.3 Hz), 169.400 (d, C(O), J=6.5 Hz), 73.121 (d, CH, J=7.7 Hz), 72.997 (d, CH, J=7.6 Hz), 71.730 (d, CH20, J=7.7 Hz), 52.502 (CH30), 52.467 (CH30), 22.968 (d, CHzCH20, J = 8.1 Hz), 22.940 (d, CH2CH20, J = 7.8 Hz), 18.846 (d, CH3CH, J=5.5 Hz), 18.573 (d, CH3CH, J=6.0 Hz), 9.629 (_C_H3CH2); fie 4.553, 3.644 (A:B, 9:11); m/e (EI) 247 (MH +, 37C1, 0.04%), 245.0345 (MH +, C7H15OsPC1 requires 245.0346, 0.43), 205 (MH + -C3H6, 37C1, 2.30), 203 (MH + -C3H6, 7.49), 187 (M + --C2H302, 37C1, 12.79), 185 IM + -C2H302, 37.69), 167 (MH + - C 3 H 7 - C I , 17.32), 145 (M + -C4H603, C1, 49.39), 143 (M + --C4H603, 100), 87 (C4H702+, 35.42), 59 (C2H302 +, 22.61).
3'-Azido-3'-deoxythymidine-5'-methyl (methyl lactyl) phosphate (5a) This was prepared by a method entirely analogous to that used for (3b) above, except that 1% methanol in chloroform was used as the chromatographic eluant. Thus, from 0.08 g of AZT was isolated 0.13 g (73%) of (5a) as a gum. 6c 170.604 (m, C(O)CH), 163.593 (C-2, A), 163.546 (C-2, B), 150.134 (C4, A), 150.120 (C-4, B), 135.179 (C-6, A), 135.136 (C-6, B), 111.607 (C-5, B), 111.484 (C-5, A), 84.621 (C-I'), 82.321 (d, C-4', A, J=8.3 Hz), 81.971 (d, C4', B, J=7.8 Hz), 72.530 (d, CHO, A, J=5.3 Hz), 72.370 (d, CHO, B, J=5.1 Hz), 66.738 (d, C-5', A, J=5.8 Hz), 66.331 (d, C-5', B, J=5.6 Hz), 60.141 (C-3', A), 59.902 (C-3', B), 55.045 (d, CH3OP, B, J= 11.8 Hz), 54.409 (d, CH3OP, A, J= 11.9 Hz), 52.719 (_c_n3oc, A), 52.665 (_C_H3OC,B), 37.605 (C-2', A), 37.489 (C-2', B), 19.082 (m, CH3CH), 12.369 (5-Me); 6p -2.442, -2.487 (A:B, 2:3); f n 8.95 (1H, s, NH), 7.426, 7.399 (1H, d, H-6, A, B, J = 1.25, 1.22 Hz), 6.24 (1H, m, H-I'), 4.97, 4.91 (1H, m, CHO, A, B), 4.523-4.245 (3H, m, H-3', H-5'), 4.02 (1H, m, H-4'), 3.877-3.731 (6H, m, CH3OP, CH3OC), 2.42 (1H, m, H-2'), 2.31 (1H, m, H-2'), 1.922, 1.916 (3H, d, 5-Me, B, A, J= 1.18, 1.22 Hz), 1.56 (3H, m, CH_H_H_~CH).
3'-Azido-3'-deoxythymidine-5'-propyl (methyl lactyl) phosphate (5b) This was prepared by a method entirely analogous to that used for (5a) above. Thus, from 0.13 g of AZT was isolated 0.33 g (73%) of (5b) as a gum. 6c 170.866 (d, C(O)CH, A, J=3.7 Hz), 170.511 (d, __C(O)CH, B, J=4.6 Hz), 163.953 (C-2, A), 163.909 (C-2, B), 150.416 (C-4, A), 150.382 (C-4, B), 135.029 (C-6, A), 134.995 (C-6, B), 111.464 (C-5, B), 111.329 (C-5, A), 84.440 (C-l'), 82.176 (d, C-4', A, J=8.2 Hz), 81.971 (d, C4', B, J=8.0 Hz), 72.205 (d, CHO, A, J= 5.2 Hz), 72.052 (d, CHO, B, J= 5.2 Hz), 70.160 (d, CH2CH20, B, J=6.1 Hz), 69.659 (d, CH2CH20, A, J=5.2 Hz), 66.566 (d, C-5', A, J=6.1 Hz), 66.185 (d, C-5', B, J=5.6 Hz), 60.160 (C-3', A), 59.909 (C-3', B), 52.514 (CH30, A), 52.441 ( c n 3 0 , B), 37.356 (C-2', A), 37.238 (C-2', B), 23.361 (m, CH2CH20), 19.972 (d, __CH3CH,B, J=5.4 Hz), 18.918 (d, CH3CH, A, J=5.6 Hz), 12.259 (5-Me), 9.765 (_C_H3CH2, A), 9.732 (_C_H3CH2, B); 6p -2.168,
206 -2.312 (A:B 4:5); 6/4 10.078, 10.030 (1H, s, NH, B, A), 7.43 (1H, M, H-6), 6.25 (1H, m, H-I'), 4.94 (1H, m, CHO), 4.36 (1H, m, H-3'), 4.32 (2H, m, H-5'), 4.103 (1H, q, H-4', J = 6.88 Hz), 4.02 (2H, m, CH2CH20), 3.739 (3H, s, CH30), 2.42 (1H, m, H-2'), 2.34 (1H, m, H-2'), 1.903, 1.896 (3H, d, 5-Me, B, A, J = 1.23 Hz, J = 1.19 Hz), 1.689 (2H, septet, CH2CH20, J = 7.01 Hz), 1.53 (3H, m, CH3CH), 0.94 (3H, m, CH3CH2); m/e (EI) 476 (MH +, 0.40%), 416 (M + -C2H302, 0.01), 373 (MH + -C4H703, 0.11), 350 (M + -thymine, 0.12), 329 (1.19), 307 (9.20), 250 (AZT.H + - H 2 0 , 2.48), 227 (1.64), 209 (MH + - A Z T ) , 185 (C4HvO3PO3H3 +, 3.44), 167 (C4HvO3PO2H +, 11.46), 126 (thymine.H +, 7.99), 125 (thymine +, 16.13), 87 (C4H702+, 3.52), 99 (C5H702+, 11.42), 81 (C5H50 +, 100).
Biological evaluation C8166 T-lymphoblastoid cells were incubated with 10 TCIDso HIV-1 (RF strain) at 37°C for 1.5 h. The cells were washed three times with phosphatebuffered saline (PBSA:Dulbecco A); aliquots of 2 × 105 cells were resuspended in 1.5 ml of growth medium in the presence of each compound at half log dilutions and incubated at 37°C for 72 h in a 95:5% air/CO2 atmosphere. HIV antigen was measured in the supernatants using a commercial ELISA following the steps described by the manufacturers (Coulter Electronics Ltd., Luton, Beds, U.K.) (Kinchington et al., 1989). AZT and ddC were used as internal controls together with uninfected and infected, untreated cells. To test for compound toxicity, aliquots of 2 × 105 uninfected cells were cultured with the compounds for 72 h. The cells were then washed with PBSA and resuspended in 200/~1 of growth medium containing ~4C protein hydrolysate. After 12 h the cells were harvested and the 14C incorporation was measured and compared with uptake observed in the untreated cells. Results
The synthetic strategy adopted for the target compounds involved the prior synthesis of the appropriate phosphorochloridate [ROC(O)CHR'O][R"O]POC1, and its reaction with a suitable nucleoside analogue. This was based on the success of this strategy for simple, and halo-substituted, alkyl phosphate derivatives (McGuigan et al., 1990a,b). Thus, ethyl glycolate was prepared by the reaction of glycolic acid and ethanol in the presence of acid catalysis (Vogel, 1964), and ethyl phosphorodichloridate was prepared by the reaction of ethanol and phosphoryl chloride in the presence of triethylamine (Kosolapoff, 1950). Ethyl (ethyl glycolyl) phosphorochloridate (la) was prepared by the reaction of these reagents in ether at low temperature. The novel product gave a single resonance in the 3Xp NMR, at 6 + 3.3. This is slightly further upfield than the signal for the starting phosphorodichloridate, as would be expected (Mark et al., 1969). Similarly prepared by the reaction of ethyl glycolate with
207 O O I) jf Et--O--C--CH2--O--P--CI OR
0 HN~ /
o
o
la lb lc ld
R=Et R=nPr R=nHex R=C12Has
Me II
o~-..~
2aR=Et 2b R=nPr ,c
nH
x
0 Me 0
0
oH'~""N~J
o.I
3a R=nPr 3b R=n-C12H2s
°" ~_2 Na
0 O II It Me--O--C--CH-O--P--CI I I Me OR
4a R=Me 4b R=nPr
0 HN~ ' ~ , Me
o II
o ii
o~.Y
I
Me--O--C--CIH-- O-- IP-- 0--~ _OJ Me OR ~
5a R=Me 5b R=nPr
Na Scheme
1.
the appropriate alkyl phosphorodichloridates were the propyl and hexyl analogues (lb,c). These phosphorochloridates were used to phosphorylate nucleosides in pyridine at ambient temperature. In the first instance, phosphate derivatives (2a-c) of 3'-acetylthymidine were prepared. This not only served as a chemical model, but was also of some biological interest. In particular, we have suggested that suitable phosphorylation of nucleosides which are inactive against, for example, HIV (such as 3'-acetylthymidine) may yield active materials by a mechanism we term 'kinase by-pass' (McGuigan et al., 1990a,b).
208
Phosphorus-31 N M R analysis of the first homologue (2a) revealed two closelyspaced signals, at 6 1.6. The multiplicity corresponds to the presence of diastereoisomers resulting from the asymmetry of the phosphorus centre. The ratio of the signals (diastereoisomers) is not unity (2:1) indicating some preference for the formation (or isolation) of one stereoisomer from the racemic phosphorochloridate reacting with the optically pure nucleoside. In the case of (2b,c) the mixtures were separated into their pure diastereoisomers by chromatography (HPLC for 2b). The roughly equi-molar yields of the two isomers, in each case, indicated no such stereoisomeric preference as noted for (2a). The 13C N M R spectra of (2a-c) are particularly informative. In the case of (2a) the presence of the isomeric mixture leads to a complex spectrum. In particular, almost every signal is split into (at least) two closely spaced resonances, indicating the non-equivalence of the carbon atoms in the two isomers; including carbons ostensibly remote from the site of the mixed stereochemistry (the phosphate). The un-equal ratio of the two isomers in (2a) is useful, as it allows un-equivocal assignment of the signals for each individual isomer. The isomeric purities of (2b-c) by comparison, are evident from the relative simplicity of the 13C spectra. For each of (2a-c), in every case, where it can be resolved, phosphorus-coupling is noted for carbon atoms within 3 bonds of the phosphorus. The magnitude of the coupling varies, from 4 Hz (C = O) to 8 Hz (C4'). Similarly, 1H N M R data are complex for the mixed isomers of (2a), but are well resolved for the purified isomers of (2b~). The compounds were also characterised by H P L C (2a-c), FAB mass spectrometry (2b-c) and microanalysis (2c). It is interesting to note that, in both cases studied by mass spectrometry, the two isomers differ significantly in their fragmentation patterns. Propyl (ethyl glycolyl) phosphorochloridate (lb) was similarly allowed to react with the anti-HIV nucleoside analogue AZT (Mitsuya et al., 1985) to yield the target compound (3a). Again, the 3 p N M R spectrum clearly reveals the presence of two isomers in this sample. However, in this case the 1:1 ratio precluded assignment of each signal to each isomer. Compound (3a) was further characterised by HPLC, mass spectrometry, and microanalysis. At this stage it was considered to be of interest to prepare a derivative where the (simple) alkyl phosphate group was a long aliphatic chain. The rationale was that this would increase the lipophilicity of the pro-drug, and enhance membrane penetration (Gouyette et al., 1989). Thus, dodecyl (ethyl glycolyl) phosphorochloridate (ld) was prepared by the reaction of dodecyl phosphorodichloridate with ethyl glycolate. Compound (ld) was isolated in high yield, and characterised by 31p and 13C N M R and mass spectrometry. In the preparation of the previous AZT derivative (3a) a very extended reaction time (14 days) had been necessary in order to achieve complete reaction. It seemed likely that the reaction of (ld) to produce (3b) would be even slower, simply on steric grounds, and alternative reaction conditions were therefore employed. In particular, we have previously found (McGuigan et al., 1990c; Curley et al.,
209 1990) that THF/N-methylimidazole conditions of Van Boom and co-workers (Van Boom et al., 1975) lead to a rapid phosphorylation with even poorlyreactive phosphoramidates. Thus, reagent (ld) was allowed to react with AZT under these conditions. This gave a complete reaction (on TLC) after only 48 h, and (3b) was subsequently isolated in high yield, as a roughly 2:1 mixture of stereoisomers. It is interesting to note that this is also the ratio observed for the ethyl phosphate of acetylthymidine (2a), whereas the propyl (2b, 3a) and hexyl phosphates (2c) were produced in roughly equal isomeric ratios. It could be that there is a steric preference for one isomer when the alkyl (phosphate) substituent is substantially smaller, or larger, than the ethyl glycolyl moiety; which might have a steric requirement similar to a pentyl or hexyl chain. The structure and purity of (3b) was also confirmed by HPLC, mass spectrometry, and microanalysis. It was of interest to probe the effect on biological activity of branching on the a-carbon of the glycolyl moiety. This is analogous to comparing various amino acids in the phosphoramidate series (Devine et al., 1990). In particular, the synthesis of a-methyl glycolates (lactates) was pursued. Thus, S-(-)-methyl lactate was allowed to react with phosphoryl chloride in the presence of triethylamine to yield 31(methyllactyl) phosphorodichloridate in moderate yield. This revealed a single P N M R signal at 6 6.5; rather down-field of that noted above for simple alkyl phosphorodichloridates. In the 13C NMR, phosphoruscoupling was noted for carbon atoms within three bonds of the phosphorus, and 1H N M R and microanalysis data were also consistent with the structure and purity of the dichloridate. On reaction with methanol, this yielded methyl (methyl lactyl) phosphorochloridate (4a) in near-quantitative yield. On account of the chirality of the starting methyl lactate, compound (4a) was a mixture of diastereoisomers about the phosphorus centre, and gave two signals in the 31p NMR. The difference in chemical shift was quite large (ca 1 ppm), but no stereoselection was noted (1:1). Many of the 13C NMR signals were similarly split, and almost all further displayed phosphorus coupling. The structure and purity of phosphorochloridate (4a) was confirmed by mass spectrometry and microanalysis. To probe the effect of alkyl (phosphate) elongation in this series, propyl (methyl lactyl) phosphorochloridate (4b) was prepared, by the reaction of propyl phosphorodichloridate with methyl lactate. Again, 31p, 13C, and 1H NMR confirmed the presence of two stereoisomers, in a l:l ratio, and mass spectral data were also recorded. Phosphorochloridate (4a) was allowed to react with AZT in THF containing N-methylimidazole to give target compound (5a) in high yield. Two diastereoisomers were apparent from the 31p NMR, with a ratio of 3:2. It is interesting that the signals were significantly further upfield than previously noted for the analogous glycolyl compounds. Many of the 13C and ~H NMR signals were also split as a result of the isomeric nature of (5a). Similarly prepared was the propyl analogue (5b). Consistent with the suggestion above concerning the variation of the diastereomeric ratios, the ratio was now nearer
210 TABLE 1 Antiviral activity of derivatives reported Compound
EDso a
TDs0
2b 2c 3a 3b 5a 5b
> 100 > 100 0.5 3 0.6 10
NT NT > 100 75 > 100 > 100
ED50 is that concentration of drug in #M which reduces viral proliferation by 50%. The TDs0 is that concentration of drug which causes 50% toxicity to uninfected cells; for full details see methods section.
1:1. Other spectroscopic data confirmed the structure and purity of (5b). The acetylthymidine compounds (2b-c) and the AZT compounds (3a-b) and (5a-b) were tested against HIV-1 in vitro, using methods we have described (Kinchington et al., 1989), the results being given in Table 1.
Discussion
The acetylthymidine compounds (2b~) were entirely inactive at the highest concentration tested (100 ~M). Thus, the aim of activation via kinase by-pass is not successful in this instance. However, the AZT derivatives (3a-b) and (5a-b) were all inhibitors of viral proliferation. In each case, the activity of the compounds decreased on lengthening the alkyl phosphate chain (3a vs. 3b; 5a vs. 5b), or on substitution of an ethyl glycolate by a methyl lactate moiety (3a vs. 5b). In the latter case a direct comparison is possible, and suggests a difference in activity in the 2 series of approximately 20-fold. The reasons for these differences remain unclear. In the first case, the lengthening of the alkyl phosphate chain was designed to improve the lipophilicity, and hence membrane penetration, of the compounds. The inverse effect on biological activity suggests either that membrane penetration is not a limiting factor for the compounds, or that other detrimental effects overwhelm any such advantage. In the case of the glycolate/lactate comparison, the difference in activity is quite major. However, just such changes in activity have been noted for phosphoramidate derivatives, where the amino acid side-chain (analogous to the site of alteration here) is modified only slightly (McGuigan et al., 1990c; McGuigan et al., 1991). In order to further probe the origins of this difference in activity, compounds (3a) and (5b) were incubated in growth medium at 37°C, and at 5°C, with controls in DMSO/water. At 37°C, both nucleotides decomposed entirely over a period of one week when dissolved in growth medium, with only 6% decomposition in DMSO/water under similar conditions. In growth medium at lower temperature, the decomposition was somewhat reduced (25% unchanged after 1 week). In the cases where decomposition was complete, HPLC revealed
211
the generation of AZT, but only in trace amounts (ca 0.05%). Thus, unless the release of AZT were greatly facilitated in vitro, it would seem likely that nucleoside release is not the primary mode of action of these compounds. On the other hand, major quantities of several very polar compounds were generated from (3a) and (5b), particularly in growth medium at 37°C. The precise structures of these hydrolysis products were not determined, but their mobility on TLC and HPLC suggests that they are free nucleotides or polar derivatives thereof. No significant differences could be noted in the hydrolysis of the two derivatives (3a) and (5b), either in a qualitative or quantitative sense. Thus no explanation of the 20-fold difference in their activities was apparent from the hydrolysis study; suggesting the involvement of some in vitro parameter in this distinction. In conclusion, we have demonstrated that phosphate triester derivatives of AZT are moderate to potent inhibitors of HIV when one of the alkyl chains is ester-containing; in particular is glycolate or lactate. Ethyl glycolate appears to be more efficacious in this regard than methyl lactate. Lengthening of the remaining alkyl phosphate group appears to lead to a reduction in biological activity. The origins of the differences in activity in the glycolate/lactate series do not appear to correspond to simple hydrolytic lability, and the mechanisms by which the compounds act remain unclear. However, the general level of activity of the ester-substituted compounds (3a-b) and (5a-b) is consistent with a mechanism of action involving intracellular delivery of the free nucleotide, or a close derivative thereof.
Acknowledgements CN thanks the SERC for a studentship. CMc thanks Mr. S. Corker (University College London) and Mr. S.J. Turner (Southampton) for HPLC analyses. DK thanks the MRC and Roche products U.K. Ltd for financial support.
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212 Gouyette, C., Neumann, J.M., Fauve, R. and Huynh-Dinh, T. (1989) 6- and l-substituted mannosyl phosphotriesters as lipophilic macrophage-targeted carriers of antiviral nucleosides. Tetrahedron Lett. 30, 6019 6022. Hunston, R.N., Jones, A.S., McGuigan, C., Walker, R.T., Balzarini, J. and DeClercq, E. (1984) Synthesis and biological properties of some cyclic phosphotriesters derived from 2'-deoxy-5fluorouridine. J, Med. Chem. 27, 440-444. Jones, B.C.N.M., McGuigan, C. and Riley, P.A. (1989) Synthesis and biological evaluation of some phosphate triester derivatives of the anti-cancer drug araC. Nucleic Acids Res. 17, 7195-7201. Kinchington, D., Galpin, S.A., O'Connor, T.J., Jeffries, D.J. and Williamson, J.D. (1989) Comparison of antigen capture assays against HIV-1 in evaluating anti-viral compounds. AIDS 3, 101-104. Kosolapoff, G.M. (1950) Organophosphorus compounds. Wiley, New York. Mark, V., Dungan, C.H., Crutchfield, M.M. and Van Wazer, J.R. (1969) Compilation of 3~p NMR data. In: M. Grayson and E.J. Griffith (Eds) Topics in Phosphorus Chemistry, vol. 5, Wiley, New York. McGuigan, C., Tollerfield, S.M. and Riley, P.A. (1989) Synthesis and biological evaluation of some phosphate triester derivatives of the anti-viral drug araA. Nucleic Acids Res. 17, 6065-6075. McGuigan, C., Nicholls, S.R., O'Connor, T.J. and Kinchington, D. (1990a) Synthesis of some novel dialkyl phosphate derivatives of 3'-modified nucleosides as potential anti-AIDS drugs. Antiviral Chem. Chemother. 1, 25 33. McGuigan, C., O'Connor, T.J., Nicholls, S.R., Nickson, C. and Kinchington, D. (1990b) Synthesis and anti-HIV activity of some novel substituted dialkyl phosphate derivatives of AZT and ddCyd. Antiviral Chem. Chemother. 1,355 360. McGuigan, C., Devine, K.G., O'Connor, T.J., Galpin, S.A., Jeffries, D.J. and Kinchington, D. (1990c) Synthesis and evaluation of some novel phosphoramidate derivatives of 3'-azido-3'deoxythymidine (AZT) as anti-HIV compounds. Antiviral Chem. Chemother. 1, 107-113. McGuigan, C., Devine, K.G., O'Connor, T.J. and Kinchington, D. (1991) Synthesis and anti-HIV activity of some haloalkyl phosphoramidate derivatives of 3'-azido-3'-deoxythymidine (AZT): potent activity of the trichloroethyl methoxyalaninyl compound. Antiviral Res. 15, 255 263. Mitsuya, H., Weinhold, K.J., Furman, P.A., St. Clair, M.H., Nusinoff-Lehrman, S., Gallo, R.C., Bolognesi, D., Barry, D.W. and Broder, S. (1985) 3'-Azido-3'-deoxythymidine(BWA509U): An agent that inhibits the infectivity and cytopathic effect of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci. USA 82, 7096-7100. Van Boom, J.H., Burgers, P.M.J., Crea, R., Luyten, W.C.M.M. and Reese, C.B. (1975) Phosphorylation of nucleoside derivatives with aryl phosphoramidochloridates. Tetrahedron 31, 2953-2959. Vogel, A.I. (1964) Practical organic chemistry. 3rd Edn, Longmans, London.
