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4958 J. Med. Chem.

1996, 39, 4958-4965

Synthesis, in Vitro Antiviral Evaluation, and Stability Studies of
Bis(S-acyl-2-thioethyl) Ester Derivatives of
9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) as Potential PMEA Prodrugs
with Improved Oral Bioavailability†

Samira Benzaria,‡,∇ Hélène Pélicano,‡ Richard Johnson,‡ Georges Maury,‡ Jean-Louis Imbach,‡
Anne-Marie Aubertin,§ Georges Obert,§,| and Gilles Gosselin*,‡
Laboratoire de Chimie Bioorganique, case courrier 008, UMR CNRS-USTL 5625, Université Montpellier II, Sciences et
Techniques du Languedoc, Place Eugène Bataillon, 34095 Montpellier Cédex 5, France, and Institut de Virologie de la Faculté
de Médecine de Strasbourg, Unité INSERM 74, 3 Rue Koeberlé, 67000 Strasbourg, France

Received April 17, 1996X

A new series of hitherto unknown 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) phospho-
nodiester derivatives incorporating carboxyesterase-labile S-acyl-2-thioethyl (SATE) moieties
as transient phosphonate-protecting groups was prepared in an attempt to increase the oral
bioavailability of the antiviral agent PMEA. We report here a direct comparison of the in
vitro anti-HIV and anti-HSV activities as well as the in vitro stability between the bis(SATE)
derivatives and the already known PMEA prodrugs, namely, bis[(pivaloyloxy)methyl (POM)]-
and bis[dithiodiethyl (DTE)]PMEA. All of the compounds tested showed an enhanced in vitro
antiviral activity compared to the parent PMEA. The bis(POM)- and bis(tBu-SATE)PMEA
derivatives were the most effective. However, striking differences between these two compounds
were found during the stability studies. In particular the bis(tBu-SATE)PMEA was found to
be more stable than bis(POM)PMEA in human gastric juice and human serum, suggesting it
could be considered as a promising candidate for further in vivo development.

9-[2-(Phosphonomethoxy)ethyl]adenine [PMEA (1);
Figure 1] has demonstrated broad spectrum antiviral
activity against human immunodeficiency virus (HIV)
and other retroviruses.1,2 PMEA is also active against
various DNA viruses, including hepatitis B virus, herpes
simplex virus (HSV), cytomegalovirus, and Epstein-Barr
virus.1,2 In addition to in vitro activity, PMEA has
demonstrated in vivo efficacy when administrated in-
travenously, intraperitoneally, or intramuscularly.1,2
Thus, PMEA is of interest both as a potential antiret-
roviral drug for HIV infections and for the treatment of
some of the opportunistic infections associated with
AIDS. It has undergone phase I/II clinical trials where
it exhibited activity against HIV in vivo.3 However, the
potential therapeutic use of PMEA could be limited by Figure 1. Structure of PMEA and its phosphonodiester
its poor oral bioavailability, which has been reported derivatives studied.
to be <1% in monkeys4 and 7.8-11% in rats.5,6 The
poor oral bioavailability is due to the phosphonate masking these charges with neutral substituents to
negative charges that are present in PMEA at physi- form more lipophilic derivatives capable of crossing the
ological pH. Therefore, the concept of temporarily gastrointestinal wall and reverting back to the parent
PMEA in plasma was attempted.
† This work is taken, in part, from the Ph.D. Dissertation of S.
Compared to prodrugs of nucleoside monophos-
Benzaria, Université de Montpellier II, June 23, 1995. It has been
phates,7-9 relatively few examples of PMEA derivatives
presented in preliminary form at the Eleventh International Round have so far been reported in the literature. One PMEA
Table on Nucleosides, Nucleotides, and their Biological Applications, prodrug has been synthesized by linking a synthetic
Sept. 7-11, 1994, Leuven, Belgium. For the proceedings, see: Ben-
zaria, S.; Gosselin, G.; Pélicano, H.; Maury, G.; Aubertin, A.-M.; Obert, polymer bearing mannosylated residues to PMEA.10
G.; Kirn, A.; Imbach, J.-L. New Prodrugs of 9-(2-phosphonomethoxy- More recently, several derivatives, including hydrogeno-
ethyl)adenine [PMEA]: synthesis and stability studies. Nucleosides phosphinate,11 mono- or bis(phosphonoamidate),6 and
Nucleotides 1995, 14, 563-565.
* Author for correspondence. Tel: (33) 4-67-14-38-55. Fax: (33) 4-67- mono- or bis(phosphonoester)6,12-16 functionalities, have
04-20-29. E-mail: been prepared as potential prodrugs of PMEA. The best
‡ Université Montpellier II.
§ Unite INSERM 74. results were obtained when PMEA was esterified with
∇ Present address: Laboratory of Medicinal Chemistry, Develop- two transient, enzyme-labile phosphonate-protecting
mental Therapeutics Program, Division of Cancer Treatment, National groups, which were later removed by a specific enzy-
Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
| Deceased on June 20, 1995. matic system. For instance, we have previously re-
X Abstract published in Advance ACS Abstracts, November 1, 1996. ported16 that the bis[dithiodiethyl (DTE)]PMEA (2;
S0022-2623(96)00289-0 CCC: $12.00 © 1996 American Chemical Society

which were prepared by reacting 2-iodoethanol diester derivatives 2. showed an increase in the in vitro anti-HIV activity of PMEA. Thus. which were performed more rapidly.17 and the bis[(pivaloyloxy)methyl (POM)]PMEA (3.SATE Derivatives as Potential PMEA Prodrugs Journal of Medicinal Chemistry.15 which consisted in yields of 75-82% after purification by silica gel of reacting PMEA with chloromethyl pivaloate in the column chromatography. 3 2 2 In our laboratory. It is noteworthy that deriva- presence of the hindered base N. 4c 0. Furthermore.4 their in vitro anti-HIV and anti-HSV activities. (ii) CH CO H/H O/MeOH. 1996. Figure 1) was examined as an efficient PMEA prodrug. 3. bis(DTE). two cell culture systems (Table 1). to identify the decomposition products from the stability reaction of 516 with iodomethyl pivaloate. c CC50.05 3.25 a(i) 5. We report herein 1 >10 >10 0. we have independently developed b.04 4. we PMEA (9a) was obtained as a byproduct (3%) during chose an approach similar to that developed in the case the detritylation of 7a. b EC50.4-triazole (MSNT) to afford the correspond. Antiviral Activity of the Phosphonodiester for the transient protection of nucleoside monophos- Derivatives 2-4 Compared to That of PMEA (1) in Two Cell phates.2.1 23 their chemical and enzymatic stabilities compared to the 4b 0. tives 4a-c could be crystallized.9 a All data represent average values for at least three separate Results and Discussion experiments.74 0. and 4a 1. 25 4959 Scheme 1. R ) Me. Dowex resin (acetate form).8 PMEA. R ) Ph. Under the assay 3-nitro-1. to allow solubilization in DMF. we acid provided the target PMEA prodrugs 4a-d as oils did not use the published procedure. (acyloxy)alkyl groups have also been studied as carboxyesterase-mediated bio- reversible groups.36 followed by studies in different media.22. 39. isolated as byproducts. d.31 The mono(Me-SATE)- For the synthesis of the title compounds 4a-d. The monoesters 8b-d were also hydroxylated derivative with a base-protected PMEA.N′-dicyclohexylmor. More importantly. 50% cytotoxic concentration (in µM) improved the overall yield and facilitated the execution or concentration required to reduce the viability of uninfected cells of some steps. compd EC50b CC50c EC50b CC50c (SATE) derivatives (4a-d.5 0.18-21 and recent studies have demonstrated that this compound improved PMEA oral bioavailability 2-fold in rats6 and 5-fold in monkeys.9 50 2 67 3 0. No.26 were condensed with their inhibitory effects on the replication of HIV-1 in 5 in pyridine in the presence of 1-mesitylene-2-sulfonyl. which is soluble in organic solvents.08 0. conditions. PMEA (1) was prepared according to operating procedures is below (10%. pyridine. and bis(POM) PMEA (3). 50% effective con- centration (in µM) or concentration required to inhibit the replica- Holy’s procedure31 with some modifications32-35 that tion of HIV-1 by 50%. Vol.26-30 Based on the favorable results obtained Lines Infected with HIV-1a with the SATE protection of various drugs.24. R ) tBu.and bis(POM).22 despite its low aqueous solubility and stability. MSNT. whose activation is mediated by reductases. PMEA (2). bis(DTE)- pholinecarboxamidine.1 7.8 0. sometimes without any preliminary purification. prepared by acid treatment of 8b-d and purified on PMEA (3). gave a 18% yield of the bis(POM). Figure 1). the carboxyesterase-labile S-acylthioethyl (SATE) group Table 1. Synthesis of Bis(SATE)PMEA Prodrugs 4a-d and Their Corresponding Monoesters 9a-da Figure 1). R ) iPr.09 13 two known PMEA prodrugs.51 3. we decided to prepare and study a series of PMEA bis. 4d 0. The hydroxythioester precursors Antiviral Activity.03 5.42 >10 the synthesis of these new PMEA prodrugs. all the tested prodrugs enhanced the in vitro . c.6 0. or by 50%.23 Phase I/II clinical trials are currently ongoing to evalu- ate its efficacy following oral administration in AIDS patients.41 2. The variation of these results under standard Chemistry. Treatment of 7a-d with acetic Concerning the synthesis of the bis(POM)PMEA (3).15 Antiretroviral activity and phar- macokinetics of orally administrated bis(POM)PMEA in mice have been reported. We opted to overcome the solubility problem by using authentic samples of the corresponding mono(SATE) the N6-monomethoxytritylated derivative (5)16 of PMEA derivatives (9a-d) of PMEA were required as standards (Scheme 1).6. in terms of MT-4 CEM-SS in vitro anti-HIV activity and preliminary stability.6. Synthesis of Bis(POM)PMEA (3) Scheme 2.4 0. PMEA (1) and the phosphono- 6a-d.6. The synthesis of bis(DTE)PMEA (2) was effected as previ. a. The monoesters 9b-d were treatment with acid.65 1. of bis(DTE)PMEA. as well as 2 6. unlike 4d. ing protected phosphonodiesters 7a-d in yields of 58- ously reported16 and involved the condensation of a 86% (Scheme 2). and 4a-d were evaluated for with the corresponding thio acid.

Therefore. noester f PMEA. step.26 on-line HPLC cleaning (POM)PMEA (3) and the bis(tBu-SATE)PMEA (4c) were method. enzyme-labile prodrugs. Liquid Chromatogr. using the recently described.01) a reference compound for this evaluation because it is aThe determination of partition coefficients was carried out by the most effective PMEA oral prodrug reported to HPLC by using a microscale method adapted from that described date. 4a > 2 ∼ 1. Measured Partition Factors (between Octanol and and it should be lipophilic enough to cross the gas- Water) of the Phosphonodiesters 2-4 Compared to That of trointestinal wall. pH 5. which represents a free- toxicity compared to the parent nucleoside (manuscript enzyme system but a nucleophile-enriched medium. J.48 0. in HSV-2-infected MRC-5 cells. such as water and pH 7. Waters) in this technique derivative 2 showed limited efficacy. The bis(POM)- that the derivatization of PMEA increases not only its PMEA (3) was the least stable under these conditions intracellular concentration but the consequent intrac. hydrolysis than the bis(DTE) derivative 2 (half-life of 5 which are the active and cytotoxic forms of the drug.2) or slightly acidic (milliQ the derivatives followed the order 3 > 4c ∼ 4b > 4d > water. as reflected of the SATE chain does not influence the reactivity of by the CC50 values (Tables 1 and 3).01) ((0..4960 Journal of Medicinal Chemistry. we do not believe nism involving nucleophilic attack at the R carbon of this to be the case. (iv) in RPMI 1640 alone. Due to the lack of authentic were also evaluated against HSV-1 and -2 in two cell samples in the case of 2 and 3. only in preparation)..5 µM for PMEA. The prodrugs appear to be serum. Table 2. We have recently published26 the . presumably due allowed the elimination of proteins before the sample to its lower lipophilicity compared to the other diesters. require an endocytosis-like process.11 0. For a designed oral prodrug to sample). (Table 5). 3. compared to >24 h for 2). enzymatic system involved in prodrug activation and giving rise to the corresponding monoesters. The in pH 7. Kelley. 25 Benzaria et al. into plasma after oral administration. 3. A rapid microscale method for the determi.31 3.01) ((0. we used an ion-pairing reagent (tetrabutylammo- We can tentatively conclude from the results pre. with EC50 values of 0. it was necessary to study their chemical and enzy- log Pa -4.2 suggest that the variation proportional enhancement of cytotoxicity. Vol. (v) in pH 2 to an increase in cellular uptake followed by intracel. in the case of PMEA. respectively. The lower EC50 values for at 37 °C (i) in water. The DTE (Guard-Pak. fied by HPLC coinjection of the corresponding mono- PMEA (1) and its phosphonodiester derivatives 2-4 (SATE) derivatives 9a-d. buffer. Indeed. It proved to be more sensitive to chemical ellular concentration of the phosphorylated anabolites. 1640 containing 10% heat-inactivated fetal calf serum pared to that of the parent molecule 1. L.. which was not Therefore. 39.21 2. and (vii) in human lular release of PMEA.1 h for 3. No. com. it appears that the apparent in simple media. unlike the PMEA. nium sulfate) in order to avoid the coelimination of the sented in Tables 1 and 2 that lipophilicity is not the polar PMEA and monoester derivatives produced after only factor that influences the antiviral activity of these degradation of the prodrugs in the various media. possible that these toxic effects are due to the release This result is in accordance with a hydrolysis mecha- of the phosphonate-protecting groups. single decomposition products for the three compounds less. Although it is this protecting group under the conditions of the assay. but it is not more potent. Merski.04) ((0.2 buffer had the same decomposition pathway. J. it must be resistant enough to any hydroly. Jr. we have recently shown that the phosphorous atom38 furthest from the acyl thioester use of SATE groups as transient phosphate protections moiety (Scheme 3).37 or the ATP mem. C. For 4a-d the prodrugs’ relative stabilities are also likely to be the decomposition products were unambiguously identi- crucial factors. and 4a were selected for testing.2 prodrug approach is not well-suited to improve the buffer. Table 4 shows that the bis(SATE) µM. we made an extrapola- lines (Table 3). These various media were thought to be valid lipophilic enough to cross the cellular membrane through in vitro models for the different types of degradation simple diffusion. The use of a reverse-phase (RP) precolumn found to be the most potent antiviral agents. A. It should be noted that every prodrug-related gain in The comparable decomposition rates obtained for antiviral potency was in all cases accompanied by a 4a-d in water and at pH 7.2 buffer. matic stabilities of the PMEA prodrugs. to test the usefulness of PMEA (1) 2 and the newly synthesized bis(SATE) derivatives 4a- 1 2 3 4a 4b 4c 4d d. 1996. Crude aliquots of incubates were directly analyzed by brane receptor1 for intracellular transport.22 previously for various nucleoside analogues (Ford. (ii) in pH 7.6. During this cleaning as reflected by their measured log P values (Table 2).5) pH. we additionally observed PMEA onstrated for the bis(POM)PMEA. Again. way: phosphonodiester derivative f phosphonomo- sis that might occur before it reaches the bloodstream.22 formation (as ascertained by coinjection with authentic Stability Studies. (vi) in human gastric juice. Neverthe. which seems to that may affect the prodrugs during oral administration. The bis(POM) ester 3 was chosen as ((0. as already dem. com- prodrugs 4a-d were the most stable toward chemical pared to 33. 14.6. the decomposi- 1991. The bis. In order to measure the relative chemical and enzy- nation of partition coefficients by HPLC.93 matic stabilities.01) ((0. For instance. H.41 3. reached the RP analytical column. presumably following the decomposition path- be effective. δ-pak C18. compound 4d is We observed that all the prodrugs tested in water and more lipophilic than 3. and 4a-d (initial concentration 5 × 10-5 M) were studied anti-HIV activity of PMEA.05) ((0.03) ((0. The inhibiting capacity of hydrolysis at neutral (pH 7. A more likely explanation would be 2.32 2. of 5’-mononucleotides does not induce any additive For the study in RPMI 1640. In culture medium. this strategy may prove useful to deliver the PMEA tested.97 compounds formed. compounds 3 and 4c proved to tion based on the retention times and UV spectra of the be the most potent. tion pathways and kinetic data for compounds 2.91 and 0. The corresponding monoesters were detected as antiviral selectivity index (ratio CC50/EC50). (iii) in RPMI the neutral phosphonodiester derivatives 2-4. can be attributed (culture medium). 3365-3386).

c Percent of decomposi.39 36.8 3. consisting of the formation of the corresponding mo- proposed mechanisms for the decomposition of SATE noester derivative as the sole product for all the pro- prodrugs and the release of the parent mononucleotide drugs tested.5 >100 29.SATE Derivatives as Potential PMEA Prodrugs Journal of Medicinal Chemistry.9 ( 2.5 ( 9. 4d ND 25. which represents an appropriate model 4b NDa 8.35 ( 0. the SATE prodrugs 4a-d in such culture media. as reflected by their half-lives in human serum (Table mains (i) carboxyesterase activity. b.7 h culture medium.6 days) and might be t1/2 attributed to the lack of reductases in the medium or compd RPMI 1640 culture medium to the poor affinity of 2 toward the reductases.2 ( 3.89 2.4 1.1 18.73 0. all the SATE prodrugs proved to be more stable than bis(POM)PMEA (3).6 >100 2 22.4 27.2%a 3%c 4c 7.c See the corresponding footnotes in Table 1.02 M) for Buffer) in Human Gastric Juice and Human Serum for the the Phosphonodiester Derivatives 2-4 Phosphonodiester Derivatives 2-4 compd water pH 7.6 8.7 days in Table 6 show a good correlation for these two media.3 ( 7. Lyon. suppose that 4a-d could be more stable than 2 and 3 tives.1 126 ( 38 12.7 days 32.2%a 1.07 ( 0.62 1.6 ( 4. The corresponding compounds were dissolved in the filtered Scheme 3.8 ( 6.8 >100 3 0.1 148 ( 27 4b 1.20 ( 0. group.75 ( 2.45 1. Calculated Rate of Hydrolysis at pH 2 (Glycine/HCl pH 5. The relative stability of 2 1640 and in Culture Medium is quite surprising (half-life 1. a ND.7 4d 2.4 ( 8.39 23 ( 11. b Human serum from healthy volunteers was a gift from the Centre Regional de Transfusion Sanguine (Montpellier.8%c 25. It is noteworthy that except for 4a.54 30.23 4.23 ( 0. 25 4961 Table 3. which would increase their absorption.5 h a Percent of decomposition after 9 days of incubation. The values reported 9d ND 2.1 4a 14. the PMEA prodrugs were 9b ND 1.4 days t1/2 <5 min 4a 13. the heat-inactivated serum added to RPMI there re. the two SATE prodrugs 4a. d Percent of decomposition after 5 days of incubation. 39.6 4c 1.3 26. 1996.9 days 13. As Table 5 clearly points out.97 12.56 ( 8.b. Cuber (Institut National de la Santé et de la Recherche Médicale. and (ii) phosphodiesterase activity.14 ( 0. pH 5. This suggests that in tion.2 h For their acid stability.5 ( 6.7 ( 7.24 1.15 ( 0.91 ( 0.43 ( 0. for which the measured half-life of 5 h compares favorably with the previously reported value. which leads to a 6). Vol.3 days t1/2 <5 min 4b 8.4 1.2 compd pH 2 human gastric juicea human serumb 2 t1/2 3.8%c t1/2 5.5) and at pH 7.89 ( 0. This last result leads us to intermediates corresponding to the mono(SATE) deriva. The half-life of 4a in culture medium was shorter in the gastric environment following oral administra- than in RPMI alone (Table 5).39 22.5 >100 32.5 ( 8. In each case.22 This result suggests that the SATE Table 5.5 10.7%d t1/2 11 min 4c 14.69 ( 0.5 ( 6.7 h 9a ND 19.7 ( 1. Table 4.4 days t1/2 4.7 ( 5. 0.7%c 3 t1/2 3.4 a All data represent average values for at least three different experiments.87 ( 0. aliquots were periodically removed and directly analyzed by (Ammonium Buffer.7 ( 10.3 h for neutral conditions where chemical and enzymatic 4c ND 3. France)] was centrifuged for 15 min at 3000 rpm at 4 °C.8 days t1/2 <5 min 3 t1/2 3.2 ( 9.6 days 3 5h 5h emerged as the most stable prodrug in media such as 4a >24 h 3.8%c 11.3) [obtained from Dr J.2 °C.3). b Percent a The human gastric juice (pH 1.2 days t1/2 2. Proposed Hydrolysis Mechanism of PMEA gastric juice up to a 50 µM concentration. are faster elimination of the first enzyme-labile SATE likely to be hydrolyzed immediately to PMEA once in . U-45. of decomposition after 26 h of incubation.9 32. as well as 2 and 3. Anti-HSV-1 and -2 Activity of the Phosphonodiester Derivatives 2-4 Compared to That of PMEA (1) in Two Infected Cell Linesa Vero MRC-5 EC50b EC50b compd HSV-1 HSV-2 CC50c HSV-1 HSV-2 CC50c 1 38.4 days hydrolysis can occur.32 15.1%d t1/2 4 h 4d 10. tion after 24 h of incubation.2 ( 2.7 ( 3.2%a 4%c 4a 20.2 (Ammonium Acetate Buffer.5) and at pH 7.6 days in human gastric juice (pH 1. No.48 ( 1.6 33. It should be noted that we proved to be more acid resistant than the already known confirmed hereby the formation of the hypothesized PMEA prodrugs 2 and 3.6%c 4b 12.9%b 2 t1/2 4.2 days evaluated not only in the usual pH 2 test buffer but also 9c ND 9.9%a 1.-C.38 48. not determined. 0.28 0. which leads to the elimination of the second SATE protecting group (we also report in Table 5 the half-life of the PMEA monoester derivatives 9a-d). c Percent of decomposition after 7 days of incubation. However.7%c 4d 9%c 5%d t1/2 1.97 ( 0.37 1.02 M) HPLC.12 ( 0. Table 6.3 ( 8. Calculated Half-Lives of the Phosphonodiester thioester might be more stable toward carboxyesterase Derivatives 2-4 and the Phosphonomonoesters 9a-d in RPMI activity than a simple ester. the bis(tBu-SATE)PMEA (4c) 2 >24 h 1. Calculated Rate of Decomposition in Water (Milli-Q.56 ( 0. for which we observed the same decomposition pathway. After incubation at 37 Prodrugs 4a-d in Water (Milli-Q. France).3 1.

J ) 7. 1H and 1H. CH2N).95 forms of administration. J ) 6.88 (t.8 Hz. 2-H and 8-H). FAB mass aromatic). a model 600E system (M .and bis.0-3. 15H. the crystalline 1H and 1H.3-6. filtered. 12 h. Moreover. controller. 2. 2 × CH3- DA 5000 mass data system.99 serum (half-life.H)-. 9H. 7.75 mg. 2 × S-CH2-CH2-O.8 (m. CH2-CH2-N).9 Hz. 12H.45. J ) 4.3-6. λmin 228 nm ( 3700). tBu-SATE derivative 4c emerged as the most promising 18H. 8H. Silica gel . 674 (M .O′-bis[(S-acetylthio)- given in δ values.4962 Journal of Medicinal Chemistry. the reaction mixture was neutralized with an aqueous solution Chemical Synthesis. FAB MS (<0.2067. Elemental analyses were performed by the Service de Mi. using the same system as indicated below in the Stability and (CH3)2CH). t. CH2-P.iPr-C(O) + H)-. rylthio)ethyl]phosphonomethoxy]ethyl}adenine (8b). D2O with a Bruker AC 250 spectrometer. under reduced pressure.8-3. absorbance detector. 7. 39. The compound to be analyzed was eluted O). 2 and 3.5. G/T) m/e at 3 kV with a total discharge current of 20 mA.49 as a ethyl]phosphonomethoxy]ethyl}adenine (7a). Iodomethyl pivaloate36 (966. 260 nm ( 12 000). circulation. The 2-H and 8-H).O′-Bis[(pivaloyloxy)methyl]phosphonomethoxy]- prodrugs (4c. G/T) Decomposition Studies section. carried out on a Waters Assoc. No. 3H. Column chromatography of the ity in neutral and acidic conditions. 2 × S-CH2-CH2-O).33 (s. the (d. 604 600E multisolvent delivery system. 7. General Procedure for the Preparation of the Phos- mine its antiretroviral efficacy after oral administration phonodiester Derivatives 7a-d and the Phosphonomo- vis-à-vis the bis(POM) derivative 3 and the parent noesters 8b-d. J ) 6. 15H. 2H. 14H cal shifts are reported relative to external H3PO4. out on a rotary evaporator under reduced pressure. Xe atoms were used for the gun C(O)-). J ) 5. (MSNT. J ) 6. 4H. 1.0-3. 2H. 2H. 25 Benzaria et al. 3 equiv) was added to a solution of 516 and the appropriate hydroxythioester 826 in anhydrous pyridine (24 Experimental Section mL/mmol of 5). residue on silica gel with a stepwise gradient of methanol (0- 3%) in dichloromethane afforded the title compound 3 as an indicates that the bis(SATE) derivatives might enhance oil (67.86 (t. (CD3)(CD2H)SO being set at δH 2. J ) 4.5. Among the SATE series. filtered. 6H. High-resolution mass butyrylthio)ethyl]phosphonomethoxy]ethyl}adenine (7b) spectra (HRMS) were obtained by using FAB+. 272 (M . (pH 7. The different retention times m/e 674 (M . NH. respectively. UV (ethanol) λmax a markedly greater chemical and enzymatic stability. 2.d) showed greater stability in human ethyl}adenine (3).9 (m. phosphorous-containing (t.90 Bruker AC 250 spectrometer with proton decoupling. compound. Silica gel chromatography [eluent. 4. S-CH2-CH2-O. 3. In vivo studies with 4c are presently ongoing to deter. CH2N). FAB MS (<0. 8H.6 (m. (2s. FAB MS(<0. and the nol (0-100%) in dichloromethane] gave 7b as a solid (86%) results were within (0.5 mg. 2 × (CH3)2CH-).32 (t. -OCH3). PMEA oral bioavailability. CH2- 100 × 4. The matrix 750 (M + H)+.4 Hz.O′-bis[(S-iso- of glycerol and thioglycerol (G/T). 1H NMR spectra were run at sodium sulfate.15 and 7. After stirring at room temperature for 12 h. 31P NMR (DMSO-d6) δ 12. 0. evaporated under reduced pressure.88 (2s. C18).27 (t. ethyl]phosphonomethoxy]ethyl}adenine (8c). -CH2-P-). J ) 6. 1H and complished by UV absorbance followed by charring with 10% 1H. column was a reverse-phase analytical column (Hypersil.12 (s. G/T) m/e 386 (M - Piv-O-CH2)-. C18.2000 that of the currently developed bis(POM)PMEA (3) with (M + H). water. S-CH2-CH2- (Guard Pak.7 (m.8 (m. and evaporated (POM)PMEA. The reaction mixture was neutralized with an and biodistribution different from (and probably better aqueous solution of 1 M triethylammonium bicarbonate buffer than) the parent PMEA. NH.H)-.70 (s.54 (d.70 (s. 1. article 9385) at atmospheric pressure. CH2-CH2-N. 14H aromatic). The residue obtained after evaporation was dissolved PMEA prodrugs 4a-d are chemically and enzymatically in chloroform (50 mL). and coevaporated with toluene and methanol. NH.7 Hz. 2H.5. 1. 2-H and 8-H). 1H. compounds were detected by spraying with Hanes molybdate CH2-P. This greater stabil. 2H. 8 mL). 31P NMR (DMSO-d6) δ 22.8-2. 2 × S-CH2-CH2-O). (CH3)2CH-). and a base line 810 data workstation. 2 × -O-CH2-). 2. CH2-CH2-N). J ) 4. 31P NMR (DMSO-d6) δ performance liquid chromatography (HPLC) studies were 22.3-6.09 (2s. and the organic layer was washed with more stable than the already known bis(DTE). Evaporation of solvents was carried of 1 M triethylammonium hydrogenocarbonate buffer (pH 7.4 reagent.08 gel 60 (Merck. G/T) m/e 806 (M + H)+. Melting 2 times the number of moles of MSNT) and extracted with points were determined with a Gallenkamp MFB-595-010-M chloroform and water. 7. 3. The crude residue Conclusion was treated with a (8:1:1. -CH2-N). 2 spectra were recorded in the positive-ion or negative-ion mode × S-CH2-CH2-O. 1-Mesitylene-2-sulfonyl-3-nitro-1. Division de Vernaison (France). 2H. a 486 tunable 8b: 1H NMR (DMSO-d6) δ 8. 1H and 1H.04 (d. 3 µm) protected by a prefilter and a precolumn P. Vol.78 mmol) in anhydrous pyridine (10 mL) and stirred at room temperature indicated that they could show in vivo bioavailability for 36 h.8-2. 2 × (CH3)2CH).28 and 7. 3. On the other hand.iPr-C(O)-S-CH2-CH2 . 3. FAB MS (>0.iPr-C(O)-S-CH2-CH2)-. 2-H and 8-H). 2 × (CH3)3C-). CH2-CH2-N).5 h). 4.14 and 8.32 ethanolic sulfuric acid with heating. 3H.90 (2s. 4H.32 (2H. water.4-triazole compound PMEA (1). 1996. dried over sodium sulfate. unit equipped with a model G/T) m/e 804 (M .7 Hz. Silica gel reference. stepwise gradient of metha- croanalyses du CNRS. Chemical shifts are N6-(4-Monomethoxytrityl)-9-{2-[O. 4. a model U6K sample injector.5.8 (m. The organic layer was dried over apparatus and are uncorrected. as well as in serum. NH2). and methanol (25 mL) and stirred at room temperature for The present results demonstrate that the bis(SATE). 6H. 31P NMR (DMSO-d6) δ 22. 15H. visualization of products being ac. J ) 6. of 4 and 1. CH2N).7 Hz. (d.2.8 Hz. N 6 -(4-Monomethoxytrityl)-9-{2-[O. 3. UV spectra and N6-(4-Monomethoxytrityl)-9-{2-[O-mono[(S-isobuty- were recorded on an Uvikon 810 (Kontron) spectrometer.01 on a JEOL DX 300 mass spectrometer operating with a JMA. 7b: 1H NMR (DMSO-d6) δ 8. v/v/v) mixture of acetic acid.7 Hz. 3.iPr-C(O)-S-CH2-CH2)-. 14H aromatic). 3. High. article 5554). stepwise gradient of methanol (0- were performed in order to confirm proton assignments. combining an antiviral potency similar to NBA) m/e 502 (M + H)+. -OCH3). calcd for C20H33N5O8P 502. HRMS 502. 18%): 1H NMR (DMSO-d6) δ 8. 2H. 544 (M . 31P 2%) in dichloromethane] gave 7a as a solid (59%) after NMR spectra were recorded at ambient temperature on a lyophilization in dioxane: 1H NMR (DMSO-d6) δ 8. TLC was after lyophilization in dioxane and the more polar 8b as a performed on precoated aluminum sheets of silica gel 60 F254 powder (8%) after lyophilization in a solution of dioxane and (Merck.2. 4.8 Hz. are also indicated in this section.O′-bis[(S-pival- The test compounds were found to be pure by rigorous oylthio)ethyl]phosphonomethoxy]ethyl}adenine (7c) and HPLC analysis. Deuterium exchange and decoupling experiments chromatography [eluent.8 (m. water.2Piv-O-CH2 + H)-. Chemi. used was 3-nitrobenzyl alcohol (NBA) or a mixture (50:50. 4.15 and 7.7 (m.01 (t. J ) form of 4a-c might facilitate the formulation of oral 12.30 (s.6 mm. high-field multinuclear NMR spectroscopy. 3. 5. FAB MS (>0. FAB MS (>0.4% of the theoretical values. v/v) N 6 -(4-Monomethoxytrityl)-9-{2-[O. N6-(4-Monomethoxytrityl)-9-{2-[O-mono[(S-pivaloylthio)- and high-resolution mass spectroscopy. which mmol) was added to a solution of 516 (544 mg. -OCH3).39 Column chromatography was carried out on silica Hz.1 Hz. the two other SATE 9-{2-[O. (t. respectively. 2H. and evaporated to dryness under ambient temperature in (CD3)2SO (DMSO-d6) or DMSO-d6 + reduced pressure. 4H. 2. 4.

mixture of water and dioxane (9:1. FAB MS (<0.8-3.16 (s. CH2-P.0-3. NBA) m/e 402 (M .8 (m.70 (s. 12H. 2 × 9-{2-[O.0 Hz.9-7. MS (>0. H. 2-H and 8-H). 2H. 2H. 7c: 1H NMR (DMSO-d6) δ 8. MS (>0. 9-{2-[O.H)-.8 (m. FAB MS (<0. (CH3)2CH-). S-CH2-CH2-O).3 Hz.8-3. 31P NMR (DMSO-d6) δ 22.4 Hz.72 (s. 22H. Dowex 1 × 2 resin (acetate . raphy [stepwise gradient of methanol (0-6%) in dichlo- 2-H and 8-H). 544 (M . 7. 3. After 12 h of stirring at room (70 mL/mol of 8).AcSCH2CH2 + H)-. CH2- 12. 31P NMR (DMSO-d6) δ 22. NH. calcd for C20H33N5O6PS2 534. NBA) m/e 562 (M + H)+. NMR (DMSO-d6) δ 19. 4a: mp 68-69 °C.6 Hz. (60%): mp 66 °C. 8-H. 2 × S-CH2-CH2-O). 31P NMR solution of dioxane and water. NBA) m/e 805 (2M . 5H. CH2-CH2-N). General Procedure for the Preparation of the Phos- phonodiester Derivatives 4a-d. N6-(4-Monomethoxytrityl)-9-{2-[O-mono[(S-benzoylthio).2iPr-C(O)-S-CH2-CH2 + H)-. Anal. 2 × S-CH2-CH2-O). 24H aromatic).1. General Procedure for the Preparation of the Phos. (C20H32N5O6PS2) C. CH2N).5-2 3.57 and 8.31 (t. 3. G/T) m/e 833 (2M - CH2-N). 4H. 4. 2-H J ) 6.18 (2s.H)-. m/e 688 (M . (0-2 M)] gave 9d (49%) as a solid after lyophilization in a methoxy]ethyl}adenine (4b). 1.11 and 8. (75%): mp 83-84 °C. 3.8 (m.4 Hz.5. 2-H and 8-H). CH2-CH2-N). 2H.Piv-S-CH2-CH2)-. 2H. 688 (M . calcd for C26H29N5O6PS2 602. (d. 3. 2H.9 (m. 4.8 (m. ethyl]phosphonomethoxy]ethyl}adenine (8d).5 Hz. 2H.99 (t.0-3. (C22H36N5O6PS2) C. 7. 6H.Bz-S-CH2-CH2)-.68 (s.7. Silica gel column chromatog. 478 (M + H)+. S-CH2-CH2-O).22 (t. DMSO-d6) δ 8.08 (q. UV -OCH3). J ) 6.17 (s. Dowex 1 × 2 resin (acetate m/e 570 (M + G + H)+.55. NH2). 2H.70 (s. 1. UV (ethanol) λmax 260 ) 6. NH2). 1H and 1H.33 (t. FAB MS (<0. S-CH2-CH2-O. 272 (M . 1996. 2. 7. ethyl}adenine (4a) and 9-{2-[O. 2 × (CH3)2CH-). UV (ethanol) λmax 260 ( 14 100). N.15 (DMSO-d6) δ 22. HRMS 562. 2 × 3. water: 1H NMR (DMSO-d6) δ 8.06 (2s. CH2N). 3. 2H. P. 2H. 244 nm ( 27 800). 2 × (CH3)2CH-).18 (2s. 231 nm ( 11 700). 7. 2. 2.0 Hz.6.Piv-S-CH2-CH2)-. 2. 2 × S-CH2-CH2-O.11 and 8. G/T) m/e form) chromatography [eluent: linear gradient of acetic acid 374 (M . 1H NMR (DMSO-d6) δ 8. 2. J + H). CH2-CH2)-.7 (m.0-3. CH2N).1316 (M 9H. 9H.7 and triethylammonium forms (10%) after lyophilization in a (m.8 Hz. J ) 6. CH2N). 8H.5 (m.O′-Mono[(S-pivaloylthio)ethyl]phosphono- (CH3)2CH-). 31P NMR (DMSO-d6) δ Hz. 2-H and 8-H). 1H and 1H.8 (m. 604 (M . G/T) m/e 708 (M . FAB MS (>0.15 (s. NH2).1595 (M + H). 1H and 1H. 2-H and 8-H).0852 (M + H).1 Hz. CH2-P). NH2).06 J ) 6. CH2-P. 39. 3. λmin 240 ( 9200). 4.4.31 (t. 8-H. 3.H)-. S-CH2-CH2-O). 1H.2Piv-S-CH2-CH2 + H)-.20 and 8.4 Hz. 3. CH2-P. 15H. CH2-P). calcd for C22H37N5O6PS2 562. 4. 18H.2.O′-Bis[(S-pivaloylthio)ethyl]phosphono- CH2-CH2 . romethane] gave 4c (76%) which was crystallized from toluene 2H. 25 4963 chromatography [eluent. 2H. the reaction mixture was evaporated under the reaction mixture was evaporated under reduced pressure reduced pressure. CH2N).Piv-S.SATE Derivatives as Potential PMEA Prodrugs Journal of Medicinal Chemistry. 4. 272 (M . NH2).57 (t. 2H. λmin 250 ( 26 500). J ) 4. J ) 6.28 (t. 6H.O′-Mono[(S-isobutyrylthio)ethyl]phosphono- ethyl]phosphonomethoxy]ethyl}adenine (9a).1923. J ) gave 4d (75%) as an oil: 1H NMR (DMSO-d6) δ 8. 4H. 31P NMR (DMSO. HRMS 602. J ) 5.0 Hz. 31P NMR (DMSO-d6) δ 22. FAB MS (<0. S-CH2. J ) 5.0 CH2-O).62 (t. J ) 6. FAB MS (<0. 4H.Piv + H)-. 4. 12H. HRMS 478. N. J ) 5. NBA) m/e oylthio)ethyl]phosphonomethoxy]ethyl}adenine (7d) and 416 (M . 3H.11 (s. appropriate fractions and purification on Dowex 1 × 2 resin 2-H and 8-H). 8H. CH2.94 (d. 4H.09 (d. 3. CH2-CH2-N). 1. Vol.89 (2s. 402 (M . 2-H. 2. 1.0- romethane] gave 4b (81%) which was crystallized from CCl4 7.9 (m. 2H. 2H. CH2N).O′-Bis[(S-acetylthio)ethyl]phosphonomethoxy]. 31P NMR (DMSO-d6) δ 22.O′-Mono[(S-acetylthio). CH2)-. 2H. 4H. 2H. G/T) m/e 436 (M . Silica gel column chromatog- 8c: 1H NMR (DMSO-d6) δ 8.8-2.55. 2 × (CH3)2CH-). -OCH3). 602 (M + H)+. Dowex 1 × 2 resin (acetate column chromatography [stepwise gradient of methanol (0.7 (m.30 (t.19 (s. and 1H. J ) 4.83 (t. CH2-CH2-N). 1H and 1H. 1H NMR (DMSO-d6) δ 8.3 Hz. (CH3)2CH-). 100%) in dichloromethane] gave 7d as a solid (58%) after N.87 (t.8 Hz. 4. and methanol methanol (75 mL/mol of 7).7 (m. J (2s. 2H. v/v/v) mixture of acetic acid. (C16H24N5O6PS2) C.20 and 8.4 Hz. CH2-P.Bz-S-CH2-CH2)-. M)]. 2-H and 8-H). G/T) methoxy]ethyl}adenine (9c). S. 2 × S-CH2-CH2-O).7 (m. and with a (8:1:1. J d6) δ 22. 3. 272 (M . 708 (M . methoxy]ethyl}adenine (4c). S. G/T) m/e 534 (M + H)+. 2 × S-CH2- (<0. HRMS 8H. 3. 230 nm ( 10 400).7 (m. 2H.09 (d. 534. (CH3)3C-C(O)-).01 (t. CH2-P. S-CH2-CH2-O.28 and 7. CH2-CH2-N. 2-H and 8-H).70 (s. FAB MS (<0. form) chromatography [eluent: linear gradient of acetic acid 6%) in dichloromethane] gave 4a (89%) which was crystallized (0-2 M)] gave 9d (46%) as a solid after lyophilization in from toluene (67%) and 9a (3%) after evaporation of the water: 1H NMR (DMSO-d6) δ 8. 832 (M .H)-. CH2N).9-3.Ac-S-CH2-CH2)-. S-CH2-CH2-O. 12 600). J ) 5.98 (t. 272 (M . 4. 223 nm ( 9800).O′-Bis[(S-benzoylthio)ethyl]phosphonomethoxy]- and triethylammonium forms (10%) after lyophilization in a ethyl}adenine (4d). 2. 4. 3H. 14H aromatic). J H)-.8. 7.23 (t. form) chromatography [eluent: linear gradient of acetic acid 9-{2-[O. 223 nm ( 10 100).3 Hz. G/T) m/e 8d: 1H NMR (DMSO-d6) δ 8. and 7.1-3.11 and 8. 2 × S-CH2-CH2-O. FAB 10 400). 31P NMR (DMSO-d6) δ 19.27 (t. 272 (M .H)-.H)-. water. H. 31P and 8-H).03 (t. FAB (<0. G/T). CH2-O). 2-H. G/T) m/e 749 (2M . CH2N). 8. J ) 8. 1.3-6. 9a: 1H NMR (D2O) δ 8. 4H.38 (2s.88 (t. 4. 4.03 (t. 3. J ) 5.4 Hz.2Bz-S-CH2-CH2 + H)-. FAB MS ( 30 900). was evaporated under reduced pressure. v/v): 1H NMR (D2O + raphy [stepwise gradient of methanol (0-6%) in dichlo. S-CH2-CH2-O). 2H. 2H.94 (d.85 (t. 4.O′-Bis[(S-isobutyrylthio)ethyl]phosphono. 2H. CH2-P. 1H and 1H. 3H. (acetate form) [eluent: linear gradient of acetic acid (0. 2 × S-CH2-CH2-O). stepwise gradient of methanol (0. 2H. v/v/v) mixture of acetic acid. 1H and 1H. No. The corresponding 7 was phonomonoesters 9b-d. G/T). 2. 231 nm ( 11 000). 2 × (CH3)3C-C(O)-). 2H. 31P NMR 9-{2-[O. 9H. CH2N). FAB MS (>0. FAB MS (<0. m/e P.4 Hz. stepwise gradient of methanol (0.0984.00 (t. CH3-C(O)-). (ethanol) λmax 260 ( 15 000).1. 2H. iPr-C(O)-S-CH2-CH2)-. 272 (M - CH2-CH2-N).46 and 8. 2 × S-CH2-CH2-O).3-6.9 Hz.07 4. ( 11 100). lyophilization in dioxane and 8d as a 3:97 mixture of the acidic 9-{2-[O. 9-{2-[O.9 Hz. 2 × S-CH2-CH2-O). [stepwise gradient of methanol (0-6%) in dichloromethane] 6. CH2-P. After 12 h of stirring at room temperature. 374 (M methoxy]ethyl}adenine (9d). water.08 (t.0-3. ) 6.9-3. CH2-CH2-N). J ) 6.8-2. 4. FAB N 6 -(4-Monomethoxytrityl)-9-{2-[O.0 Hz. 1.11 and 8. 7. 1H and 1H. CH2N). 4H. Silica gel column chromatography solution of dioxane and water. S-CH2-CH2-O. 4H.5.9 (m. J ) 4. 10H. m/e 874 (M + H)+. 2H. 136 (BH2)+. Anal. J ) 6.97 (t. CH2-CH2-N).18 (s. J ) 6.2 Hz.34 (s.Piv-S.O′-bis[(S-benz. J ) 4. temperature.8. CH2N). 2 × S-CH2-CH2-O. Silica gel methoxy]ethyl}adenine (9b). 7d: 1H NMR (DMSO-d6) δ 8.8 (m. and extracted with water and chloroform. S-CH2-CH2-O. 2H. 2. 2.34 (t. 4H.0 Hz. CH2-N).06 (2s.1610. m/e 834 (M + H)+. 31P NMR (DMSO-d6) δ 12.19 (s. CH2-P). 3.10 (q. -OCH3). 7. J ) 5. Silica gel UV (ethanol) λmax 260 ( 16 100).7 (m. J ) 6. CH2-P.7 Hz. 223 nm ( 10 400).6 Hz. 3.89 (2s.1297. 4. NH2).6. 1H and 1H. CH2-P. 7. G/T) m/e 872 (M . CH2- lyophilization in dioxane and 8c as a 3:97 mixture of the acidic CH2-N). 2-H and 8-H).31 (t. 2H. FAB MS ) 5.7 (m.0 Hz.3-6. 4.O′-Mono[(S-benzoylthio)ethyl]phosphono- (D2O) δ 17.H)-. J ) 6.iPr-C(O)-S- 14H aromatic). ) 8. 7.09 (2s. 2H. 2 × (CH3)3C-C(O)-). 2H.9 Hz. CH2N). 2H. NH.32 (t. 4.14 (s. 1H NMR (DMSO-d6) δ 8. -OCH3). 3. FAB MS (<0. 2 Ph). 4.5 Hz. CH2-CH2)-. NH. 4. Anal. 1H CH2-N).31 (t. 223 nm ( (<0. 1H and 1H.H)-. 1. H. 6H. FAB MS (>0.8 (m. G/T). FAB MS (>0. G/T). 27H. S-CH2-CH2-O. calcd (0-2 M)] gave 9c (54%) as a solid after lyophilization in for C16H25N5O6PS2 478. P. 544 (M .44 (2s.H)-. 8H. Ph). 2 × S-CH2-CH2-O. J ) 100%) in dichloromethane] gave 7c as a solid (58%) after 5. 4.3 . (CH3)3C-C(O)-). 416 (M .26 (t.Bz-S-CH2- 19H aromatic). CH2. 6H.1876 (M + H).Piv-S-CH2-CH2)-. S. λmin 240 ( 18H. NH. 15H. λmin 242 chromatography [eluent. 4. 3H. 3. The corresponding 8 was treated treated with a (8:1:1. 3. 6H. 2H. J ) 6. The aqueous layer 9-{2-[O. S-CH2-CH2-O).

monophosphates through a reductase-mediated activation pro- initial concentration of 2. and a linear gradient from eluent acyclic nucleoside phosphonates. M. 65. 53-55. 36. Hitchock. C. Y. Germany).-L. Chem.41 For CEM cells. The 1995. Naesens. J. Gosselin. Vol.. pp 147-172. ethyl)adenine (PMEA). and Associa- The origin of the viruses and the techniques used for measur. (16) Puech.0. S 10. De Clercq. De Clercq. A. C. eluent A 2.40. serum (FBS).. 37. 1994. 9d. eluent C (v/v/v). DC.. 2127- that is. E. Bischofberger..... G. Jr. Tortolani. C. M. The 50% effective concentra. M. M. Com- ments on nucleotide delivery forms. The viral cytopathic effect (CPE) was recorded daily. Collect. 1993.. E. 963-972. Lefebvre. J.. J. 2. essential medium (MEM) supplemented with 5% fetal bovine (6) Starrett. Balzarini. 27.. Mansuri. virus activity of phosphonylmethoxyalkylpurines and -pyrim- tion (EC50) was derived from the computer-generated median idines. Antiviral Res. Abstract M.) and ARC antiviral activity of the pronucleotides was based on a reduc.2. The elution system PMEA. G/T) m/e 873 (2M .. Then. Yanachkov.4964 Journal of Medicinal Chemistry. These investigations were sup- MS (<0. Synthesis. 5. F. 1853-1869. JAI Press Inc.. Antiviral (7) Alexander. Bischofberger. 267-273. about 3 × 103 cells (approximately 30% confluent). Balzarini. H.-L. Slachmuylders. M... Yanachkova. (PMEA).. The anti-HSV assays Phosphonylmethoxyethyl)adenine (PMEA) effectively inhibits were performed by a method adapted from that described by retrovirus replication in vitro and simian immunodeficiency De Clercq. Minireview: nucleotides prodrugs. microtiter plates..41 Cells.H)-. (5) Bronson. Chem. after virus adsorption. (H. the cleaning (13) Glazier. 1994.1.. injected into the precolumn and eluted with eluent A during (17) Srinivas. ammonium acetate and acetic acid pro analysis (Merck. Antiviral Res. I. M. 13. R.. D. G. FAB Acknowledgment. 9a. Czech. J. Jaffe. in MT-4 cells. Czech.7. Schmidt tion of HIV-1-IIIB-induced cytopathogenicity. France). of different concentrations of test compounds for 5 days before (2) De Clercq. L. 3. The crude sample (80 µL..16. AIDS 1991. the determination of the of us are particularly grateful to ANRS (S.. N. Kirn. acidified 2-propanol was added to each Res. When cells (10) Midoux. M. 1).0 M. France). J. Eragny.. Vol. Chem.. M. 32. Masur. Virol. Faloon. B to eluent C programmed over 30 min was used. cytoxicity of the test compounds was measured after an Markowitz. Agence Nationale de Biological Methods. 1989. C.. water 40.. J.. Chem. Mansuri. 2. ethyl)adenine.. R. Neyts. Saint Quentin. A phase I/II study of incubation of 5 days in their presence using the colorimetric PMEA in HIV infected patients. Toxicity was considered to occur if the monolayer remained Czech. 1. 22. Biochem.P. M. colorimetric MTT assay.. Martin.. Prodrugs of analogs of nucleic acid activity was expressed as EC50 (50% effective concentration). Albrecht for excellent assistance in performing activity of the cells being measured by the property of the anti-HIV-1 assays. previously described HPLC procedure. 23. Ghazzouli. Commun. Kovacs. M.43 In this assay.45 The herpes simplex virus type 1 [HSV-1. 2. unbound particles were elimi. In vivo anti-retrovirus and anti-cytomegalovirus activity of 9-((2-phosphonylmethoxy)- human embryonic fibroblasts (cell line MRC-5).. (4) Balzarini.. oral bioavailability determination. H. A. 3 µm. D. Pharmacol.: Greenwich. The cells were grown in Eagle’s minimum 74. Polis. Antiviral Res.. C. Bethesda. calculated using the program mentioned above. A306. retention times (min) were 1.5. and in vitro evaluation of prodrugs for 1 h at 37 °C and immediately thereafter exposed to various of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine concentrations of the test compounds (from 1 mM to 1 µM).44.) for a fellowship. J. 59. phosphonate prodrug: bis(pivaloyloxymethyl)9-(2-phosphonyl- ammonium sulfate [one bottle of PIC A reagent (Waters) in methoxyethyl)adenine. 26.8. 20.-L... tion pour la Recherche sur le Cancer (ARC. 22. P. Holy. Gong.3. 16. 1993.. acetonitrile 50. MD) for critical reading of the thiazol-2-yl)-2.. A. E. J..5 mM buffered tetrabutyl. Roche. J.H)-. E. Niphuis. Preparation of 9-(2- less than 50% confluent. A276. 5 min. 19. K. Two ing inhibition of virus multiplication have previously been described. were References respectively incubated with 50 or 100 TCID50 of viruses during (1) For a review. E. L... A. A. P. Whiterock. 30 min. We are indebted to Dr. 1870-1878. V.9.. E. incubation at 37 °C. H. Girardet. 59. Montreal. M. S-CH2-CH2-O). the bis- 4b. Schellekens. E. Recherches sur le SIDA (ANRS. J.. H. Lane. A. Med.. E. E. 1996.1.P.. Fridland. Med. P. H.5-dimethyl. Antiviral efficacy in mice of oral bis(POM)-PMEA.. 9c.. Potent topical anti-herpes activity of a lipophilic precolumn was a Guard-Pak insert (Delta-Pak C18 100 Å) in phosphorus prodrug for the antiviral agent PMEA. J. 22. R. Ghaffar. 3. Synthesis and in vitro evaluation of a Darmstadt. 436 (M . 26. pH 6. Russel.. 1994.. 1990.. (pivaloyloxymethyl) ester prodrug of 9-(2-phosphonylmethoxy- mono(DTE)PMEA. We warmly thank S.. 1044-1049. 4. well. Rosenberg. J. Buckheit. Negre. (3) Walker. Aubertin. 2. a Guard-Pak holder (Waters.. V. E. Cundy.16 Briefly. 147-159. J. I. Biophys. After a 3 h bond 9-(2-phosphonylmethoxyethyl)adenine.16. 33. W. M. A. in the control wells (without drug) reached confluence. 1992. Ebeling. I.. . A. Drug cytotoxicity was determined using the quantitative 1996..5-diphenyltetrazolium bromide (MTT) into for. R. J. 1994.. CT. J.. Russel. phosphonomethoxyethyl)adenine esters as potential prodrugs. Drug targeting: anti-HSV-1 activity of mannosylated polymer- mL of MTT (5 mg/mL) was added to each well. the concentration of compound required to reduce the 2165. 1st National MTT test. In Advances in antiviral control virus-infected cell cultures. A. 9b.. F. Antiviral Res. B. components. Antiviral Res. No.. J. 23 (Suppl. Robbins. 23. 42. 37. MT-4 and CEM-SS. strain virus infection in rhesus monkeys. Int. J... Jr. 1-17... Kern.. Mayer. Pivaloyloxymethyl esters of to the column was activated. 39. M. 20. activity associated with the virus particle released in the culture supernatant. and cells were cultured in the presence 1994. I. Imbach. France). (12) Alexander. E. 167. (14) Glazier. E. Biochem.B. N. Commun. E. J. Commun. Canada. M. Stability and Decomposition Studies. Synthesis of 9-(2- determined using a 96-well plate photometer at 540 nm.. Hz. Vogel. of the test compounds were added to 96-well plates containing Antiviral Res.43 The 50% cytotoxic concentration (CC50) is the Conference on Human Retroviruses and Related Infections. different dilutions (9) Jones. Wright. Martin. J. S. M. Therapeutic potential of PMEA as an antiviral drug. Marquez (NIH. 59. Abstract 522.8. 1991. D. Abstracts of Papers. J. 21-28. S. 4. phosphinomethoxyethyl)adenine and related compounds. R. from (15) Starrett. 31P NMR (D2O + DMSO-d6) δ 16. Hitchcock. strain G (ATCC VR-734)] were propagated on Vero cells and Klunk. 4d. mono(POM)PMEA.. analytical column used was a Hypersil C18. De Clercq. Commun. 25 Benzaria et al.3. Holy. Balzarini. A. 5th International All assays were carried out on confluent cells in 96-well Conference on AIDS.. Hitchock. Masojidkova. Connely. 1995. 155-174. France). Paar. water 90.42 In parallel experiments.8. A.. the production of virus HIV-LAI was measured by quantification of the reverse transcriptase this manuscript is also greatly appreciated. R. Imbach. eluent B (v/v). L. R. L. Gosselin. R. Tortolani. 9-(2- Anti-HSV Assays on Cell Culture.C. 1995. N. N. J.. Martin. R. manuscript.. Abstracts of Papers. The assistance of G. J. ported by grants from the CNRS. was prepared as follows: stock solution S..6.1. the metabolic and G. 4c. C. T. 1994. 16. viral CPE by 50% when it had reached completion in the (8) Périgaud. Anti-HIV Assays on Cell Culture.. C.. Collect. Victor mitochondrial dehydrogenases to reduce 3-(4. J. A. Hanrahan in typing mazan. The (18) Naesens. A. De Clercq. S. P.. see: Naesens.0 L of water]. Holy. J. 120 Å. J. Broad-spectrum anti-DNA virus and anti-retro- virus production determination.. The optical density in each well of the plate was (11) Alexander.. J. Antiviral News 1994. Holy. nated by two washes. J. Pompon. 1857-1864. Ed.. drug design. Masojidkova. L. 0. D. Davey. 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