SYNTHESIS AND ANTIVIRAL EVALUATION OF 9-(S)-[3-ALKOXY-2-(PHOSPHONOMETHOXY)PROPYL]NUCLEOSIDE ALKOXYALKYL ESTERS: INHIBITORS OF HEPATITIS C VIRUS AND HIV-1 REPLICATION
We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a HCV replicons with EC50 values of 1-2 \xce\xbcM and a CC50 of>150 \xce\xbcM. Analogs with substitutions at the 3\xe2\x80\xb2-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC50 <0.01 nanomolar and a selectivity index of>4.4 million.
1. Introduction<!>2.1 Chemistry<!>2.2 Biological Activity<!>3. Conclusions<!>4.1 General Chemistry Methods<!>4.2 General procedure A. Synthesis of 3-alkoxy-2-hydroxypropyl nucleoside analogs<!>4.2.1. (S)-9-[(3-methoxy-2-hydroxy)propyl]adenine (2)<!>4.2.2. (R)-9-[(3-methoxy-2-hydroxy)propyl]adenine (3)<!>4.2.3. (R,S)-9-[(3-ethoxy-2-hydroxy)propyl]adenine (4)<!>4.2.4. (R,S)-9-[(3-isopropoxy-2-hydroxy)propyl]adenine (5)<!>4.3. General procedure B. Synthesis of 9-[(3-alkoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (6-9)<!>4.3.1. (S)-9-[(3-methoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (6)<!>4.3.2. (R)-9-[(3-methoxy-2-hydroxy)propyl] N6-monomethoxytrityladenine (7)<!>4.3.3. (R,S)-9-[(3-ethoxy-2-hydroxy)propyl] N6-monomethoxytrityladenine (8)<!>4.3.4. (R,S)-9-[(3-isopropoxy-2-hydroxy)propyl] N6-monomethoxytrityladenine (9)<!>4.4. General procedure C. Alkylation of 9-[(3-alkoxy-2-hydroxy)propyl] derivatives (6-9, 20-22, 27-29, 38, 41-42) with alkoxyalkyl p-toluenesulfonyloxymethylphosphonate. Synthesis of (10-14, 23-26, 30-33, 39, 43-44)<!>4.4.1. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl] N6-monomethoxytrityladenine, sodium salt (10)<!>4.4.2. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphonomethoxy)propyl] N6-monomethoxytrityladenine, sodium salt (11)<!>4.4.3. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl] N6-monomethoxytrityladenine, sodium salt (12)<!>4.4.4. Hexadecyloxypropyl (R,S)-9-[(3-ethoxy-2-phosphonomethoxy)propyl] N6-monomethoxytrityladenine, sodium salt (13)<!>4.4.5. Hexadecyloxypropyl (R,S)-9-[(3-isopropoxy-2-phosphonomethoxy)propyl] N6-monomethoxytrityladenine, sodium salt (13)<!>4.5. General procedure D. Synthesis of Alkoxyalkyl-9-[(3-alkoxy-2-phosphonomethoxy)propyl]adenine (15-19)<!>4.5.1. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]adenine, sodium salt (ODE-(S)-MPMPA) (15)<!>4.5.2. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphonomethoxy)propyl]adenine, sodium salt (ODE-(R)-MPMPA) (16)<!>4.5.3. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphonomethoxy)propyl]adenine, sodium salt (HDP-(S)-MPMPA) (17)<!>4.5.4. Hexadecyloxyethyl (R,S)-9-[(3-ethoxy-2-phosphonomethoxy)propyl]adenine, sodium salt (HDP-(R,S)-EPMPA) (18)<!>4.5.5. Hexadecyloxyethyl (S)-9-[(3-isopropoxy-2-phosphonomethoxy)propyl]adenine, sodium salt (HDP-(R,S)-IPPMPA) (19)<!>4.6.1 (S)-9-[(3-methoxy-2-hydroxy)propyl]-2,6-diaminopurine (20)<!>4.6.2. (R)-9-[(3-methoxy-2-hydroxy)propyl]-2,6-diaminopurine (21)<!>4.6.3. (R,S)-9-[(3-ethoxy-2-hydroxy)propyl]-2,6-diaminopurine (22)<!>4.6.4. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]2,6-diaminopurine, sodium salt (ODE-(S)-MPMPDAP) (23)<!>4.6.5. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphonomethoxy)propyl]2,6-diaminopurine, sodium salt (ODE-(R)-MPMPDAP) (24)<!>4.6.6. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphonomethoxy)propyl]-2,6-diaminopurine, sodium salt (HDP-(S)-MPMPDAP) (25)<!>4.6.7. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphonomethoxy)propyl-2,6-diaminopurine, sodium salt (HDP-(R,S)-EPMPDAP) (26)<!>4.7.1. (S)-9-[(3-methoxy-2-hydroxy)propyl]-6-O-benzylguanine (27)<!>4.7.2. (R)-9-[(3-methoxy-2-hydroxy)propyl] -6-O-benzylguanine (28)<!>4.7.3. (R,S)-9-[(3-ethoxy-2-hydroxy)propyl]-6-O-benzylguanine (29)<!>4.7.4. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]-6-O-benzylguanine (30)<!>4.7.5. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphonomethoxy)propyl]-6-O-benzylguanine (31)<!>4.7.6. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphonomethoxy)propyl]-6-O-benzylguanine (32)<!>4.7.7. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphonomethoxy)propyl]-6-O-benzylguanine (33)<!>4.7.8 General procedure E. Synthesis of Alkoxyalkyl-9-[(3-alkoxy-2-phosphonomethoxy)propyl]guanine (34-37)<!>4.7.9. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]guanine (ODE-(S)-MPMPG) (34)<!>4.7.10. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphonomethoxy)propyl]guanine (ODE- (R)-MPMPG) (35)<!>4.7.11. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphonomethoxy)propyl]guanine (HDP-(S)-MPMPG) (36)<!>4.7.12. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphonomethoxy)propyl]guanine (HDP-(R,S)-EPMPG) (37)<!>4.8.1. (S)-1-[(3-methoxy-2-hydroxy)propyl]-N4-monomethoxytritylcytosine (38)<!>4.8.2. Octadecyloxyethyl (S)-1-[(3-methoxy-2-phosphonomethoxy)propyl]-N4-monomethoxytritylcytosine (39)<!>4.8.3. Octadecyloxyethyl (S)-1-[(3-methoxy-2-phosphonomethoxy)propyl]cytosine, sodium salt (40) (ODE-(S)-MPMPC)<!>4.9.1. (S)-9-[(3-methoxy-2-hydroxy)propyl]-6-methoxypurine (41)<!>4.9.2. (S)-9-[(3-methoxy-2-hydroxy)propyl]-6-O-methylguanine (42)<!>4.9.3. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]-6-methoxypurine (HDP-(S)-MPMPMP) (43)<!>4.9.4. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphonomethoxy)propyl]-6-O-methylguanine (HDP-(S)-MPMPOMG) (44)<!>4.10.1. HCV antiviral activity<!>4.10.2. HIV-1 inhibition assays
<p>Treatment of hepatitis C virus (HCV) infection remains an important unmet medical need due to the inadequacies of current interferon-based therapy.1 In the United States there are 3 to 4 million persons with chronic HCV infection.2 A number of agents are currently in clinical development for HCV including NS3 protease inhibitors, NS5A inhibitors3 and NS5B antiviral nucleosides and non-nucleoside polymerase inhibitors.4 Clinical and in vitro data indicate that resistance develops readily with protease and non-nucleoside polymerase inhibitors.5-7 In contrast, nucleoside inhibitors, which target the catalytic site of the NS5B RNA dependent RNA polymerase, are active across different HCV genotypes and exhibit a higher barrier to resistance.8</p><p>Acyclic nucleoside phosphonates (ANPs) are an important group of broad spectrum antiviral agents with activity against double stranded DNA (dsDNA) viruses,9 or viruses which rely on reverse transcription such as HBV10 and HIV-1.11 We previously reported that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (ODE-(S)-HPMPA, 1) exhibited antiviral activity against genotype 1b and 2a HCV replicons, the first report of significant antiviral activity of an acyclic nucleoside phosphonate against an RNA virus.12 However, ODE-(S)-HPMPA has substantial cytotoxicity.12-13 To identify additional anti-HCV compounds with less cytotoxicity, we synthesized several closely related analogs having methyl, ethyl or isopropyl substitutions at the 3′-hydroxyl, octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine (ODE-(S)-MPMPA, 15), octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine (ODE-(R)-MPMPA, 16), hexadecyloxypropyl 9-(S)-[3-methoxy-2-(phosphono-methoxy)propyl]adenine (HDP-(S)-MPMPA, 17), hexadecyloxypropyl 9-(R,S)-[3-ethoxy-2-(phosphonomethoxy)propyl]adenine (HDP-(R,S)-EPMPA, 18) and hexadecyloxypropyl 9-(R,S)-[3-isopropoxy-2-(phosphonomethoxy)propyl]-adenine (HDP-(R,S)-IPPMPA, 19) (Scheme 1). We also prepared the HDP- or ODE- esters of (R and S)-MPMP-2,6 diaminopurine (23-26) (Scheme 2), guanine (34-37) (Scheme 3), cytosine (40), 6-methoxypurine (43) (Scheme 4) and 6-O-methylguanine (44) (Scheme 5). The antiviral activity of these compounds against HCV, HIV-1 and other viruses was compared with ODE-(S)-HPMPA (1).</p><!><p>Synthesis of the new ANP analogs was carried out using the synthon approach we developed previously for alkoxyalkyl esters of (S)-9-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine [(S)-HPMPA].13 As detailed in Scheme 1, adenine reacted with various alkyl glycidyl ethers under basic conditions to give a series of 9-(3-alkoxy-2-hydroxypropyl)adenines (2-5). After protection of the amino group with monomethoxytrityl, compounds 6-9 could be alkylated with octadecyloxyethyl or hexadecyloxypropyl p-toluenesulfonyloxymethyl-phosphonate, then deprotected to provide adenine derivatives 15-19. As shown in Scheme 2, 3-methoxy- and 3-ethoxy-2-phosphonomethoxypropyl analogs of 2,6-diaminopurine (23 – 26) were prepared using a similar strategy. In this case, acceptable yields (22 – 50%) of the target compounds were obtained without protection of the amino groups. Synthesis of the related guanine (Scheme 3, 34 – 37) and cytosine (Scheme 4, 40) analogs started from 6-O-benzylguanine and N4-monomethoxytritylcytosine, respectively. Methoxypurine analogs (43-44) were prepared as shown in Scheme 5.</p><!><p>Compounds were tested for anti-HCV activity in genotype 1b and 2a replicons as previously described14 and their activity compared with that of ODE-(S)-HPMPA (Table 1). ODE-(S)-MPMPA (15) retained full activity against genotype 1b and 2a replicons with EC50 values of 1.43±0.38 and 2.38±1.09 μM while the (R) isomer (16) was slightly less active with EC50 of 4.65 and 5.33 μM. HDP-(S)-MPMPA (17) was slightly less active than the corresponding ODE ester with EC50 of 2.36 (1b) and 4.64 μM (2a). When ethyl or isopropyl substitutions were made at the 3′-hydroxyl instead of methyl, the anti-HCV activity dropped slightly with HDP-(R,S)-EPMPA (18) to EC50 of 7.59 (1b) and 8.87 μM (2a) suggesting that larger substitutions are not favored. Cytotoxicity of ODE-(S)-MPMPA was substantially lower than that observed with ODE-(S)-HPMPA, CC50 >150 versus 35.6 μM. Of the various adenine analogs, ODE-(S)-MPMPA had the greatest selectivity index, >105 with genotype 1b and >63 with genotype 2a replicons. The anti-HCV activity of ODE-(S)-MPMPA is similar to that of 4′-azidocytidine and 2′-C-methylcytidine, EC50 1.13 to 1.28 μM15, and less than that of PSI-352938, a cyclic phosphate prodrug of β-D-2′-α-fluoro-2′-β-C-methylguanosine, EC50 0.13 to 0.20 μM. 16</p><p>We also prepared alkoxyalkyl MPMP esters of cytosine, guanine, 2,6-diaminopurine, 6-methoxypurine and 6-O-methylguanine. Among them, the most active anti-HCV compound was ODE-(S)-MPMPG (34) with EC50 values of 8.26 and 10.7 μM against genotype 1b and 2a, respectively; the (R) isomer (35) was slightly less active with EC50 of 12.6 and 12.4 μM. These compounds also had low cytotoxicity with CC50 values >150 μM. HDP-(S)-MPMPMP (43) also exhibited significant activity in the 8.9 to 12.5 μM range. HDP-(S)-MPMPOMG (44) and the ODE and HDP esters of both (R) and (S)-MPMPDAP (23-25) were less active with EC50 values ranging from 18 to 26 μM while ODE-(S)-MPMPC (40) was inactive.</p><p>We also evaluated the compounds in MT-2 cells infected with HIV-1 (Table 2). ODE-(S)-HPMPA (1) was highly active with an EC50 of 0.0001μM. However, the CC50 was 0.033 μM making it the most cytotoxic compound in the series. ODE-(S)-MPMPA (15) retained substantial antiviral activity with an EC50 of 0.03 μM and a CC50 of 22 μM (selectivity index 733). By way of comparison, our previous studies with adefovir and tenofovir in HIV-1 infected MT-2 cells showed EC50 values for these compounds of 1.1 and 0.65 μM, respectively. 17,18 HDP-(S)-MPMPA (17) was less active and ODE-(R)-MPMPA (16) was considerably less active and selective. Introduction of an ethoxy or isopropoxy at the 3′-hydroxyl position of the acyclic moiety (compounds 18, 19) resulted in a loss of antiviral activity (Table 2). Surprisingly, the most active compound was ODE-(R)-MPMPG (35), EC50 <1×10-5 μM and a selectivity index of >4.4 million. Interestingly, the (S) isomer (34) was substantially less active with an EC50 of 0.2 μM. The same pattern was observed with ODE-(R)-MPMPDAP (24) which was more active (EC50 = 0.4 μM) than the (S) isomer (23). As noted before with the adenine analogs, the HDP esters (25, 36) were less active. Again, introduction of larger ethyl groups at the 3′-hydroxyl of the acyclic chain of these compounds (26, 37) caused a large loss of anti-HIV activity. ODE-(S)-MPMPC (40) was less potent (EC50 = 12.7 μM) and HDP-(S)-MPMPMP (43) and HDP-(S)-MPMPOMG (44) were considerably less potent against HIV (EC50 >10μM).</p><p>ODE-(S)-MPMPA was also tested against HCMV and HSV-1 using our previous methods.19 We reported previously that ODE-(S)-HPMPA is a powerful inhibitor of the replication of orthopoxviruses, including variola,20 vaccinia and cowpox,21 and ectromelia,22 as well as other dsDNA viruses including human cytomegalovirus (HCMV) and herpes simplex virus, type 1 (HSV-1).13 We examined the effect of blocking the 3′-hydroxyl of HPMPA with 3′-methoxy on the compound's antiviral activity against dsDNA viruses including vaccinia, cowpox, HCMV and HSV-1 (Table 3). As we reported previously, ODE-(S)-HPMPA had potent antiviral activity against these viruses with EC50 values ranging from <0.1 to 20 nanomolar. However, ODE-(S)-MPMPA (15) exhibited a dramatic loss of antiviral activity with EC50 values >400 to >45 million times higher than those of ODE-(S)-HPMPA (Table 3). We reported previously that inhibition of vaccinia virus replication occurs by a unique mechanism in which (S)-HPMPA diphosphate is incorporated into DNA by the viral E9L polymerase. However, the vaccinia polymerase cannot copy across the drug lesion in HPMPA containing templates.23 The EC50 for vaccinia inhibition by ODE-(S)-HPMPA is 20 nanomolar versus 18,300 nanomolar for ODE-(S)-MPMPA, a reduction of 915-fold (Table 3). These findings generally support the principal mechanism which we described previously because incorporation of HPMPA into viral DNA, blocking further copying of the drug-containing chain is not possible with ODE-(S)-MPMPA, and any residual antiviral activity with the latter compound is due to obligatory chain termination. Presumably this is also the mechanism of action in cowpox which has a closely related DNA polymerase. The antiviral mechanism of action of HPMPA diphosphate has not been studied extensively for HCMV and HSV-1.</p><!><p>Prior studies identified ODE-(S)-HPMPA (1) as a compound with good antiviral activity against HCV.12 However, this compound has significant toxicity both in vitro and in mice, in vivo. Surprisingly, replacement of the acyclic chain hydroxyl with a methoxy group greatly reduces toxicity while anti-HCV activity is preserved in the 1-2 μM range. Selectivity indexes for ODE-(S)-MPMPA (15) range from >63 to >105, a marked improvement over the selectivity of ODE-(S)-HPMPA (1). Interestingly, blocking the 3′-hydroxyl of ODE-(S)-HPMPA with a methyl results in a marked loss of antiviral activity against double stranded DNA viruses, including vaccinia, cowpox, HCMV and HSV-1. The ODE-MPMP- analogs of guanine, 6-methoxypurine and 6-O-methylguanine were also active against HCV while the ODE (S) and (R) isomers of MPMPDAP were somewhat less potent. ODE-(S)-MPMPC was inactive. ODE-(R)-MPMPG (35) showed striking antiviral activity against HIV-1 with an EC50 of <0.01 nanomolar and a selectivity index of >4.4 million. Our results support further evaluation of ODE-(S)-MPMPA (15) for use against chronic HCV infection and ODE-(R)-MPMPG (35) for HIV infection.</p><!><p>All reagents were of commercial quality and used without further purification unless indicated otherwise. Chromatographic purification was done using the flash method with silica gel 60 (EMD Chemicals, Inc., 230–400 mesh). 1H NMR spectra were recorded on Varian HG spectrophotometers operating at 400 MHz and are reported in units of parts per million (ppm) relative to internal tetramethylsilane at 0.00 ppm. Assignments of 1H NMR signals are made using the numbering system shown in Scheme 1. Routine electrospray ionization mass spectra (ESI-MS) were recorded on a Finnigan LCQDECA spectrometer, and high resolution mass spectra (HRMS) on an Agilent 6230 Accurate-Mass TOFMS mass spectrometer in ESI negative mode. Both spectrometers are located at the small molecule facility, Department of Chemistry, University of California, San Diego and operated by Dr. Yongxuan Su. Purity of the target compounds was characterized by high performance liquid chromatography (HPLC) using a Beckman Coulter System Gold chromatography system. The analytical column was Phenomenex Synergi™ Polar-RP (4.6 × 150 mm) equipped with a SecurityGuard™ protection column. Mobile phase A was 95% water/5% methanol and mobile phase B was 95 % methanol/5% water. At a flow rate of 0.8 mL/min, gradient elution was as follows: 10 % B (0 – 3 min.); 10 % to 95 % B (3 – 20 min.); 95 % B (20 – 25 min.); 95 % to 10 % B (25 – 34 min.). Compounds were detected by ultraviolet light (UV) absorption at 274 nm. Purity of compounds was also assessed by thin layer chromatography (TLC) using Analtech silica gel-GF (250 μm) plates and the solvent system: CHCl3/MeOH/con NH4OH/H2O (70:30:3:3 v/v). TLC results were visualized with UV light, phospray (Supelco, Bellefonte, PA, USA) and charring at 400 °C. The key synthons, hexadecyloxypropyl- and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate, were prepared as described previously.13</p><!><p>The preparation of various 3-alkoxy-2-hydroxypropyl nucleoside analogs was accomplished using the base catalyzed ring-opening reaction of alkyl glycidyl ethers with nucleobases as described by Brodfuehrer et al.24 Sodium hydride (2 mmol) was added to a solution of the nucleic acid base (10 mmol) and an alkyl glycidyl ether (10 mmol) in anhydrous N,N-dimethylformamide (50 mL) and the mixture was stirred and heated to 100 °C for 6 hours. After cooling, the reaction was quenched with H2O, the solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel. Elution of the column with 10% MeOH/CH2Cl2 gave the product.</p><!><p>was synthesized from adenine and (S)-methyl glycidyl ether (TCI America, Portland, OR). 75% yield. 1H NMR (CDCl3/methanol-d4) δ 8.25 (s, 1H); 8.03 (s, 1H); 4.39-4,44 (m, 1H); 4.19-4-25 (m, 1H); 4.08-4.18 (m, 1H); 3.37-3.44 (m, 2H); 3.40 (s, 3H). MS (ESI): 224.14 [M+H]+.</p><!><p>was synthesized from adenine and (R)-methyl glycidyl ether (TCI America, Portland, OR). 72% yield. 1H NMR (methanol-d4) δ 8.20 (s, 1H, H-8); 8.08 (s, 1H, H-2); 4.39 (dd, 1H, H-1′a, J1′a,2′ = 3.4 Hz, Jgem = 14.2 Hz); 4.20 (dd, 1H, H-1′b, J1′b,2′ = 8.0 Hz, Jgem = 14.2 Hz); 4.11 (m, 1H, H-2′); 3.42 (d, 2H, H-3′, J3′,2′ = 5.2 Hz); 3.37 (s, 3H, -OCH3).</p><!><p>was synthesized from adenine and (R,S)-ethyl glycidyl ether (TCI America) with 59% yield. 1H NMR (CDCl3/methanol-d4), δ:8.24 (s, 1H); 8.04 (s, 1H); 4.40-4.65 (m, 1H); 4.20-4-27 (m, 1H); 4.10-4.15 (m, 1H); 3.55-3.57 (m, 2H); 3.46-3.54 (m, 2H); 1.21 (t, J=7Hz, 3H). MS (ESI): 238.09 [M+H]+.</p><!><p>was synthesized from adenine and (R,S) isopropyl glycidyl ether (Aldrich Chem.). 33.8% yield. 1H NMR (CDCl3/methanol-d4), δ 8.21 (s, 1H, H-8); 8.08 (s, 1H, H-2); 4.41 (dd, 1H, H-1′a, J1′a,2′ = 3.8, Jgem = 14.2 Hz); 4.23 (dd, 1H, H-1′b, J1′b,2′ = 7.6 Hz, Jgem = 14.4 Hz); 4.10 (m, 1H, H-2′); 3.60 (septet, 1H, -CH(CH3)2, J = 6.0 Hz); 1.16 (d, 6H, -CH(CH3)2).</p><!><p>The monomethoxytrityl group was used to block the exocylic amino group of adenine and was introduced by the transient protection method described by Ti et al.25 Bromotrimethylsilane (6.3 mmol) was added dropwise to a suspension of 9-[(2-hydroxy-3-alkoxy)propyl]adenine (2-5) (2.8 mmol) in dry pyridine (10 mL). The mixture was stirred 15 min. until it became clear, then monomethoxytrityl chloride (0.99 g, 3.2 mmol) and 4-(dimethylamino)pyridine (20 mg, 0.2 mmol) were added and stirring was continued overnight. The mixture was cooled with an ice bath and H2O (1 mL) was added. Stirring was continued 10 min., then con. NH4OH (1 mL) was added and the reaction was stirred 30 additional min. The mixture was allowed to warm to room temperature and filtered through a pad of celite. The filtrate was evaporated in vacuo and the residue was purified by flash column chromatography on silica gel. Gradient elution (100% hexanes to 100% ethyl acetate) afforded the N6-monomethoxytrityl 9-[(2-alkoxy-3-methoxy)propyl]adenines (6-9).</p><!><p>was synthesized from 2. 98 % yield. 1H NMR (CDCl3/methanol-d4), δ 8.17 (s, 1H, H-8); 8.13 (s, 1H, H-2); 7.39-7.75 (m, 14H, trityl); 4.50-4.59 (m, 1H); 4.31-4.39 (m, 1H); 4.22-4.30 (m, 1H); 3.45 (s, 3H); 3.50-3.60 (m, 2H); 3.55 (s, 3H). MS (ESI): 496.06 [M+H]+, 518.13 [M+Na]+.</p><!><p>was synthesized from 3. 19 % yield. 1H NMR (CDCl3/methanol-d4), δ 8.03 (s, 1H, H-8); 7.78 (s, 1H, H-2); 7.35-7.33 (m, 4H, trityl); 7.29-7.24 (m, 10H, trityl); 4.61 (d, 1H, H-1′a, J1′a,2′ = 4 Hz); 4.39 (dd, 1H, H-1′b, J1′b,2′ = 2.2 Hz, Jgem = 13.8 Hz); 4.16 (m, 1H, H-2′); 3.78 (s, 3H, Ar-OCH3); 3.41 (dd, 1H, H-3′a, J3′a,2′ = 5.2 Hz, Jgem = 9.2 Hz); 3.36 (s, 3H, CH2-OCH3); 3.33 (dd, 1H, H-3′b, J3′b,2′ = 6.2 Hz, Jgem = 9.8 Hz).</p><!><p>was synthesized from 4. 78 % yield. 1H NMR (CDCl3/methanol-d4), δ 7.98 (s, 1H); 7.25-7.34 (m, 14H); 6.82 (s, 1H); 4.35-4.43 (m, 1H); 4.15-4.24 (m, 1H); 4.05-4.15 (m, 1H); 3.78 (s, 3H); 3.52-3.56 (m, 2H); 3.44-3.47 (m, 2H); 1.20 (t, J=7Hz, 3H). MS (ESI): 509.78 [M+H]+.</p><!><p>was synthesized from 5. 32 % yield. 1H NMR (DMSO-d6) δ 8.10 (s, 1H, H-8); 7.89 (s,1H, H-2); 7.28-7.26 (m, 10H, trityl); 7.21-7.18 (m, 4H, trityl); 6.83 (d, 1H, -NH-); 5.16 (d, 1H, -OH); 4.23 (dd, 1H, H-1′a, J1′a,2′ = 3.4 Hz, Jgem = 13.8 Hz); 3.98 (dd, 1H, H-1′b, J1′b,2′ = 8.4 Hz, Jgem = 13.6 Hz); 3.92 (m, 1H, H-2′); 3.70 (s, 3H, Ar-OCH3); 3.50 (septet, 1H, -CH(CH3)2); 3.35 (dd, 1H, J3′a,2′ = 4.8 Hz, Jgem = 10 Hz); 3.27 (dd, 1H, H-3′b, J3′b,2′ = 6, Jgem = 9.6); 1.04 (dd, 6H, - CH(CH3)2).</p><!><p>Sodium t-butoxide (0.19 g, 2.0 mmol) was added to a solution of the 3-alkoxy-2-hydroxypropyl nucleoside (1.0 mmol) and the alkoxyalkyl p-toluenesulfonyloxymethylphosphonate12 (2.0 mmol) in anhydrous N,N-DMF (20 mL). The mixture was heated to 80 °C and kept overnight. After cooling, the solvents were evaporated in vacuo and the residue was purified by flash column chromatography on silica gel. The column was eluted with a gradient: chloroform 100% - chloroform-methanol (20%) to give the products.</p><!><p>was synthesized from (S)-9-[(3-methoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (6) and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate. 60% yield. 1H NMR (CDCl3/methanol-d4), δ 8.18 (s, 1H); 7.98 (s, 1H); 7.22-7.55 (m, 14H); 4.38-4.50 (m, 2H); 4.12-4.37 (m, 2H); 4.00-4.08 (m, 1H); 3.82- 3.98 (m, 2H); 3.79 (s, 3H); 3.58-3.65 (m, 2H); 3.44-3.48 (m, 2H); 3.38-3.43 (m, 2H); 3.35 (s, 3H); 1.40-1.60 (m, 2H); 1.16-1.38 (m, 30H); 0.88 (t, J = 7Hz, 3H). MS (ESI): 886.48 [M+H]+.</p><!><p>was synthesized from (R)-9-[(3-methoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (7) and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate. 43% yield. 1H NMR (CDCl3/methanol-d4), δ 8.20 (s, 1H); 7.98 (s, 1H); 7.22-7.37 (m, 14H); 4.42-4.50 (m, 1H); 4.28-4.37 (m, 1H); 3.91-3.98 (m, 2H); 3.82- 3.90 (m, 2H); 3.79 (s, 3H); 3.60-3.69 (m, 1H); 3.48-3.58 (m, 3H); 3.39-3.46 (m, 2H); 3.35 (s, 3H); 1.45-1.60 (m, 2H); 1.20-1.38 (m, 30H); 0.88 (t, J=7Hz, 3H). MS (ESI): 886.57 [M+H]+.</p><!><p>was synthesized from (S)-9-[(3-methoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (6) and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate. 77% yield. 1H NMR (CDCl3/methanol-d4), δ: 8.17 (s, 1H); 7.94 (s, 1H); 7.22-7.36 (m, 14H); 4.38-4.50 (m, 2H); 4.28-4.37 (m, 2H); 3.82- 3.98 (m, 2H); 3.79 (s, 3H); 3.58-3.65 (m, 1H); 3.38-3.58 (m, 6H); 3.34 (s, 3H); 1.78-1.87 (m, 2H); 1.44-1.60 (m, 2H); 1.10-1.40 (m, 26H); 0.88 (t, J=7Hz, 3H). MS (ESI): 870.33 [M-H]-.</p><!><p>was synthesized from (R,S)-9-[(3-ethoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (8) and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate. 80% yield. 1H NMR (CDCl3/methanol-d4), δ: 8.19 (s, 1H); 7.98 (s, 1H); 7.20-7.36 (m, 14H); 4.42-4.65 (m, 1H); 4.28-4.37 (m, 1H); 3.80- 3.95 (m, 3H); 3.78 (s, 3H); 3.48-3.65 (m, 6H); 3.28-3.48 (m, 2H); 1.78-1.87 (m, 2H); 1.44-1.55 (m, 2H); 1.08-1.30 (m, 26H); 1.15 (t, J=7Hz, 3H); 0.88 (t, J=7Hz, 3H). MS (EI): 886.42 (M+H)+.</p><!><p>was synthesized from (R,S)-9-[(3-isopropoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (9) and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate. 25% yield. 1H NMR (CDCl3/methanol-d4) δ 8.15 (s, 1H, H-8); 7.88 (s, 1H, H-2); 7.30 – 7.25 (m, 4H, trityl); 7.20 – 7.12 (m, 10H, trityl); 4.66 (dd, 1H, H-1′a, J1′a,2′ = 3.5 Hz, Jgem = 14.2 Hz); 4.47 (dd, 1H, H-1′b, J1′b2′ = 6.2 Hz, Jgem = 14.0 Hz); 4.01 (m, 2H, -P-O-CH2-); 3.88 (dd, 1H, -CHa-P-, JP,CHa = 9.2 Hz, Jgem = 13.6 Hz); 3.71 (dd, 1H, - CHb-P-, JP,CHb = 9.6 Hz, Jgem = 14.0 Hz); 3.75 – 3.55 (m, 3H, H-3′ + H-2′); 3.51 (t, 2H, -CH2-O-CH2-); 3.45 (t, 2H, -CH2-O-CH2-); 1.83 (pentet, 2H, -O-CH2CH2CH2O-); 1.53 (m, 2H, - CH2(CH2)15-); 1.27 (m, 26H, -(CH2)15-); 1.10 (d, 6H, -CH(CH3)2); 0.89 (t, 3H, -CH3).</p><!><p>Alkoxyalkyl 9-[(3-alkoxy-2-hydroxy)propyl]-N6-monomethoxytrityladenine (10-14) (0.60 mmol) was added to 80% acetic acid, stirred and heated to 60 °C for 2 hours. After cooling, the solvent was removed in vacuo and the residue purified by flash column chromatography on silica gel. Elution with 20% MeOH/ CH2Cl2 gave the products.</p><!><p>Deprotection of 10 (procedure D) gave 15 in 73% yield as a white powder. 1H NMR (CDCl3/methanol-d4) δ 8.35 (s, 1H, H-8); 8.22 (s, 1H, H-2); 4.53 (dd, H, H-1′a, J1′a2′ = 3.3 Hz, Jgem 14.3 Hz); 4.37 (dd, 1H, H-1′b, J1′b2′ = 6.6 Hz, Jgem 14.7 Hz); 4.01-3.98 (m, 3H, P-O-CH2- + H-2′); 3.87 (dd, 1H, -CHa-P-, JP,CHa = 9.2 Hz, Jgem = 13.2); 3.70 (dd, 1H, -CHb-P-, JP,CHb = 9.0 Hz, Jgem = 13.0 Hz); 3.57 (t, 2H, -CH2-O-); 3.44 (t, 2H, -O-CH2-); 1.53 (m, 2H, -O-CH2CH2(CH2)15-); 1.26 (m, 30H, -(CH2)15CH3); 0.89 (t, 3H, -CH3, J = 7Hz). MS (ESI+): 614.41 [M+H]+; HRMS (ESI-) calcd. for C 30H55N5O6P [M-H] 612.3895, found 612.3897 (E = 0.3 ppm). HPLC analysis: retention time 22.35 min., purity 96.12 %.</p><!><p>Deprotection of 11 gave 16 in 72% yield. 1H NMR (CDCl3/methanol-d4), δ: 8.24 (s, 1H); 8.21 (s, 1H); 4.43-4.54 (s, 1H); 4.25-4.35 (m, 1H); 3.88-3.98 (m, 3H); 3.80-3.88 (m, 1H); 3.50-3.60 (m, 4H); 3.38-3.48 (m, 3H); 3.37 (s, 3H); 1.49-1.56 (m, 2H); 1.20-1.35 (m, 30H); 0.88 (t, J=7Hz, 3H). MS (ESI+): 614.55 [M+H]+, 636.46 [M+Na]+. HRMS (ESI-) calcd. for C30H55N5O6P [M-H]- 612.3895, found 612.3900 (E = 0.8 ppm). HPLC analysis: retention time 21.82 min., purity 95.24 %.</p><!><p>Deprotection of 12 gave 17 in 77% yield. 1H NMR (CDCl3/methanol-d4), δ 8.28 (s, 1H); 8.23 (s, 1H); 4.48-4.61 (s, 2H); 4.32-4.37 (m, 2H); 3.91-3.96 (m, 2H); 3.80-3.86 (m, 1H); 3.58-3.64 (m,1H); 3.41-3.57 (m, 3H); 3.30-3.41 (m, 2H); 3.38 (s, 3H); 1.82-1.90 (m, 2H); 1.49-1.55 (m, 2H); 1.18-1.38 (m, 26H); 0.89 (t, J=7Hz, 3H). MS (ESI-): 598.29 [M-H]-. HRMS (ESI-) calcd. for C29H53N5O6P [M-H]- 598.3739, found 598.3737 (E = -0.3 ppm). HPLC analysis: retention time 22.43 min., purity 93.0 %.</p><!><p>Deprotection of 13 gave 18 in 92% yield. 1H NMR (CDCl3/methanol-d4), δ 8.28 (s, 1H); 8.21 (s, 1H); 4.48-4.53 (s, 1H); 4.34-4.39 (m, 1H); 3.90-4.00 (m, 3H); 3.80-3.86 (m, 1H); 3.58-3.64 (m,1H); 3.44-3.58 (m, 6H); 3.35-3.41 (m, 2H); 1.82-1.90 (m, 2H); 1.49-1.58 (m, 2H); 1.22-1.38 (m, 26H); 1.20 (t, J=7Hz, 3H); 0.89 (t, J=7Hz, 3H). MS (ESI): 612.44 (M-H)-. HRMS (ESI-) calcd. for C30H55N5O6P [M-H]- 612.3895, found 612.3898 (E = 0.5 ppm). HPLC analysis: retention time 22.60 min., purity 91.8 %</p><!><p>Deprotection of 14 gave 19 in 75% yield. 1H NMR (methanol-d4) δ 8.35 (s,1H, H-8), 8.25 (s, 1H, H-2), 4.53, (dd, 1H, H-1′a, J1′a2′ = 3.4 Hz, Jgem = 14.6 Hz), 4.39 (dd, 1H, H-1′b, J1′b2′ = 6.6 Hz, Jgem = 14.6 Hz), 3.93 (t, 2H, P-O-CHa, J = 6.8 Hz), 3.91 (t, 1H, P-O-CHb, J = 6.4Hz); 3.85 (dd, -CHa-P-, JP,CHa = 9.0 Hz, Jgem = 13 Hz), 3.66 (dd, 1H, -CHb-P-, JP,Hb = 9.6 Hz, Jgem = 13.2 Hz); 3.59-3.48 (m, 3H, H-3′ + H-2′), 3.46 (t, 2H, -CH2-O CH2), 3.37 (t, 2H, -CH2-O-CH2-), 1.80 (pentet, 2H, -O-CH2-CH2-CH2-O-), 1.51 (m, 2H, -O-CH2- CH2-(CH2)13-), 1.27 (m, 26H, -(CH2)13-), 1.13 (d, 6H, -CH(CH3)2), 0.89 (t, 3H, -CH3); MS (ESI-): 626.69 [M-H]-. HRMS (ESI-) calcd. for C31H57N5O6P [M-H]- 626.4052, found 626.4053 (E = 0.2 ppm). HPLC analysis: retention time 21.95 min., purity 97.1 %.</p><!><p>was synthesized from 2,6-diaminopurine (TCI America) and (S)-methyl glycidyl ether (procedure A). 27% yield (0.65 g). 1H NMR (CDCl3/methanol-d4), δ 7.68 (s, 1H, H-8); 4.20-4.30 (m, 1H); 4.05-4.12 (m, 2H); 3.32-3.47 (m, 2H); 3.39 (s, 3H).</p><!><p>was synthesized from 2,6-diaminopurine and (R)-methyl glycidyl ether (procedure A). 41% yield. 1H NMR (CDCl3/methanol-d4), δ 7.73 (s, 1H, H-8); 4.22 (d, 1H, H-1′a, Jgem = 12.4 Hz), 4.06-4.02 (m, 2H, H-1′b + H-2′); 3.39 (d, 2H, H-3′, J = 3.2 Hz); 3.36 (s, 3H, -OCH3).</p><!><p>was synthesized from 2,6-diaminopurine and (R,S)-ethyl glycidyl ether (procedure A). 71 % yield. 1H NMR (CDCl3/methanol-d4) δ 7.77 (s, 1H, H-8); 4.22 (dd, 1H, J1′a,2′ = 3.6 Hz, Jgem = 12.4 Hz, H-1′a); 4.06-4.02 (m, 2H, H-1′b + H-2′); 3.88 (q, 2H, -OCH2CH3); 3.39 (d, 2H, H-3′, J = 3.2 Hz); 1.16 (t, 3H, -OCH2CH3).</p><!><p>was synthesized (procedure C) from (S)-9-[(3-methoxy-2- hydroxy)propyl]2,6-diaminopurine and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate. 50% yield. 1H NMR (CDCl3/methanol-d4), δ 7.72 (s, 1H); 4.00-4.20 (m, 5H); 3.80-3.90 (m, 1H); 3.58-3.65 (m, 2H); 3.50-3.58 (m, 2H); 3.40-3.50 (m, 1H); 3.44 (s, 3H); 3.30-3.38 (m, 2H); 1.50-1.60 (m, 2H); 1.18-1.38 (m, 30H); 0.88 (t, J=7Hz, 3H). MS (ESI): 627.48 [M-H]-, 629.47 [M+H]+. HRMS (ESI-) calcd. for C30H56N6O6P [M-H]- 627.4004, found 627.4007 (E = 0.5 ppm). HPLC analysis: retention time 23.67 min., purity 91.9 %.</p><!><p>was synthesized (procedure C)from (R)-9-[(3-methoxy-2- hydroxy)propyl]2,6-diaminopurine and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate with 49% yield. 1H NMR (CDCl3/methanol-d4), δ: 7.75 (s, 1H);4.42-4.51 (m, 1H); 4.00-4.20 (m, 4H); 3.80-3.90 (m, 1H); 3.60-3.65 (m, 2H); 3.50-3.58 (m, 2H); 3.40-3.50 (m, 1H) 3.49 (s, 3H); 3.30-3.38 (m, 2H); 1.50-1.62 (m, 2H); 1.20-1.38 (m, 30H); 0.88 (t, J=7Hz, 3H). MS (ESI+): 629.55 [M+H]+. HRMS (ESI-) calcd. for C30H56N6O6P [M-H]- 627.4004, found 627.4007 (E = 0.5 ppm). HPLC analysis: retention time 23.67, purity 98.4 %.</p><!><p>was synthesized (procedure C) from (S)-9-[(3-methoxy-2- hydroxy)propyl]2,6-diaminopurine and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate with 30% yield. 1H NMR (CDCl3/methanol-d4), δ: 7.72 (s, 1H); 4.02-4.20 (m, 3H); 3.95-4.02 (m, 2H); 3.78-3.85 (m, 2H); 3.45-3.60 (m, 3H); 3.38-3.45 (m,6H); 1.82-1.95 (m, 2H); 1.45-1.60 (m, 2H); 1.20-1.38 (m, 26H); 0.88 (t, J=7Hz, 3H). MS (ESI+): 615.50 [M+H]+, 637.45 [M+Na]+. HRMS (ESI-) calcd. for C29H54N6O6P [M-H]- 613.3848, found 613.3854 (E = 1.0 ppm). HPLC analysis: retention time 22.98 min., purity 90.3%.</p><!><p>was synthesized (procedure C) from (R,S)-9-[(3-ethoxy-2- hydroxy)propyl]2,6-diaminopurine and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate with 22% yield. 1H NMR (CDCl3/methanol-d4), δ: 7.40 (s, 1H); 4.44-4.52 (m, 1H); 4.18-2.28(m, 1H); 3.91-4.10 (m, 3H); 3.80-3.90 (m, 1H); 3.45-3.60 (m, 5H); 3.38-3.45 (m, 4H); 1.82-1.95 (m, 2H); 1.45-1.60 (m, 2H); 1.15-1.38 (m, 28H); 0.88 (t, J=7Hz, 3H). MS (ESI-): 627.53 [M-H]-. HRMS (ESI-) calcd. for C30H56N6O6P [M-H]- 627.4004, found 627.4008 (E = 0.6 ppm). HPLC analysis: retention time 23.37 min., purity 96.4 %.</p><!><p>was synthesized from 6-O-benzylguanine (APAC Pharmaceutical LLC, Columbia, MD) and (S)-methyl glycidyl ether (procedure A). 49% yield. 1H NMR (CDCl3/methanol-d4), δ 7.75 (s, 1H); 7.49-7.51 (m, 2H); 7.29-7.40 (m, 3H); 5.55 (s, 2H); 4.23-4.32 (m, 1H); 4.05-4.14 (m, 2H); 3.39-3.41 (m, 2H); 3.39 (s, 3H).</p><!><p>was synthesized from 6-O-benzylguanine and (R)-methyl glycidyl ether (procedure A). 44% yield. 1H NMR (CDCl3/methanol-d4), δ 7.74 (s, 1H); 7.47-7.51 (m, 2H); 7.29-7.40 (m, 3H); 5.55 (s, 2H); 4.40-4.60 (m, 1H); 4.05-4.14 (m, 2H); 3.32-3.45 (m, 2H); 3.39 (s, 3H).</p><!><p>was synthesized from 6-O-benzylguanine and (R,S) ethyl glycidyl ether (procedure A). 51% yield. 1H NMR (CDCl3/methanol-d4), δ 7.76 (s, 1H); 7.50-7.52 (m, 2H); 7.32-7.40 (m, 3H); 5.52 (s, 2H); 4.17-4.21 (m, 1H); 3.95-4.00 (m, 2H); 3.44-3.50 (m, 2H); 3.34-3.38 (m, 2H); 1.16 (t, J=7Hz, 3H).</p><!><p>was synthesized (procedure C) from (S)-9-[(3-methoxy-2- hydroxy)propyl]-6-O-benzylguanine and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate. 26% yield. 1H NMR (CDCl3/methanol-d4), δ 7.93 (s, 1H); 7.50-7.56 (m, 2H); 7.31-7.40 (m, 3H); 5.55 (s, 2H); 4.24-4.36 (m, 1H); 3.93-4.22 (m, 1H); 3.75-3.98 (m, 4H); 3.60-3.70 (m, 4H); 3.30-3.60 (m, 8H); 1.42-1.60 (m, 2H); 1.18-1.38 (m, 30H); 0.89 (t, J=7Hz, 3H). MS (ESI): 720.51 [M+H]+.</p><!><p>was synthesized (procedure C) from (R)-9-[(3-methoxy-2- hydroxy)propyl]-6-O-benzylguanine and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate. 83% yield. 1H NMR (CDCl3/methanol-d4), δ 7.93 (s, 1H); 7.52-7.47 (m, 2H); 7.23-7.38 (m, 3H); 5.55 (s, 2H); 4.18-4.38 (m, 2H); 3.75-3.98 (m, 4H); 3.55-3.65 (m, 1H); 3.43-3.50 (m, 3H); 3.30-3.43 (m, 8H); 1.45-1.60 (m, 2H); 1.18-1.38 (m, 30H); 0.89 (t, J=7Hz, 3H). MS (ESI): 718.54 [M-H]-.</p><!><p>was synthesized (procedure C) from (S)-9-[(3-methoxy-2- hydroxy)propyl]-6-O-benzylguanine and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate. 71% yield. 1H NMR (CDCl3/methanol-d4), δ:7.94 (s, 1H); 7.49-7.55 (m, 2H); 7.24-7.40 (m, 3H); 5.55 (s, 2H);4.30-4.40 (m, 1H); 4.17-4.22 (m, 1H); 3.80- 3.92 (m, 3H); 3.72-3.92 (m, 1H); 3.55-3.62 (m, 1H); 3.40-3.52 (m, 4H); 3.28-3.40 (m, 2H); 3.37 (s, 3H); 1.75-1.85 (m, 2H); 1.44-1.60 (m, 2H); 1.16-1.38 (m, 26H); 0.89 (t, J=7Hz, 3H). MS (ESI): 706.50 [M+H]+.</p><!><p>was synthesized (procedure C) from (R,S)-9-[(3-methoxy-2-hydroxy)propyl]- 6-O-benzylguanine and hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate. 42% yield). 1H NMR (CDCl3/methanol-d4), δ 7.95 (s, 1H); 7.48-7.52 (m, 2H); 7.30-7.40 (m, 3H); 5.56 (s, 2H);4.34-4.40 (m, 1H); 4.19-4.26 (m, 1H); 3.77- 3.93 (m, 4H); 3.58-3.66 (m, 1H); 3.47-3.55 (m, 5H); 3.35-3.45 (m, 3H); 1.78-1.85 (m, 2H); 1.48-1.55 (m, 2H); 1.17-1.28 (m, 29H); 0.89 (t, J = 7 Hz, 3H). MS (ESI): 718.46 [M-H]-.</p><!><p>The protected guanine compounds (30-33) (0.71 mmol) were added to 10% trifluoroacetic acid/CH2Cl2 and the mixture was stirred at room temperature for 2 days. The solvent was removed in vacuo and the residue purified by flash column chromatography on silica gel. The column was eluted with 20% MeOH/CH2Cl2 and the crude products were recrystallized from water to give the alkoxyalkyl 9-[(3-alkoxy-2-phosphonomethoxy)propyl]guanine derivatives (34-37).</p><!><p>Deprotection of 30 gave 34 as a white powder. 93% yield. 1H NMR (CDCl3/methanol-d4), 7.82 (s, 1H); 4.24-4.36 (m, 1H); 4.10-4.28 (m, 1H); 3.95-4.05 (m, 2H); 3.78-3.90 (m, 2H); 3.62-3.73 (m, 1H); 3.52-3.60 (m, 2H); 3.40-3.50 (m, 2H); 3.25-3.40 (m, 3H); 1.45-1.60 (m, 2H); 1.18-1.38 (m, 30H); 0.89 (t, J = 7Hz, 3H). MS (ESI): 628.44 [M-H]-. HRMS (ESI-) calcd. for C30H55N5O7P [M-H]- 628.3845, found 628.3846 (E = 0.2 ppm). HPLC analysis: retention time 21.10, purity 96.2 %.</p><!><p>Deprotection of 31 gave 35 as a white powder. 67% yield. 1H NMR (CDCl3/methanol-d4), 8.07 (s, 1H, H-8); 7.51 (s, 2H, -NH2); 4.34 (dd, 1H, H-1′a, J1′a2′ = 3.9 Hz, Jgem = 14.6 Hz); 4.13 (dd, 1H, H-1′b, J1′b2′ = 6.4 Hz, Jgem = 14.3 Hz); 4.00 (m, 2H, -P-O-CH2-); 3.87 (dd, 1H, -CHa-P-, JP,CHa = 8.7 Hz, Jgem = 12.9 Hz); 3.68 (dd, 1H, -CHb-P-, JP,CHb = 9.6 Hz, Jgem = 12.8Hz); 3.59 (t, 2H, -CH2-O-CH2); 3.46 (d + t, 4H, -CH2-O-CH2 + H-3′); 3.38 (s, 3H, - OCH3); 1.50-1.62 (m, 2H, -O-CH2CH2(CH2)15-); 1.27 (m, 30H, -(CH2)15-); 0.89 (t, J = 7 Hz, 3H, -CH3). MS (ESI+): 630.29 [M+H]+; HRMS (ESI-) calcd. for C30H55N5O7P [M-H]- 628.3845, found 628.3843 (E = -0.3 ppm). HPLC analysis: retention time 22.33 min., purity 92.9 %.</p><!><p>Deprotection of 32 gave 36 as a white powder. 93% yield. 1H NMR (CDCl3/methanol-d4), 7.83 (s, 1H); 4.26-4.31 (m, 1H); 4.06-4.11 (m, 1H); 3.93- 3.98 (m, 2H); 3.81-3.86 (m, 2H); 3.60-3.63 (m, 2H); 3.48-3.57 (m, 3H); 3.35-3.44 (m, 2H); 3.37 (s, 3H); 1.84-1.89 (m, 2H); 1.52-1.58 (m, 2H); 1.15-1.40 (m, 26H); 0.88 (t, J=7Hz, 3H). MS (ESI): 616.45 [M+H]+, 638.40 [M+Na]+. HRMS (ESI-) calcd. for C29H53N5O7P [M-H]- 614.3688, found 614.3687 (E = -0.2 ppm). HPLC analysis: retention time 20.55 min., purity 93.6 %.</p><!><p>Deprotection of 33 gave 37. 88% yield. 1H NMR (CDCl3/methanol-d4), 7.85 (s, 1H); 4.28-4.33 (m, 1H); 4.11-4.17 (m, 1H); 3.92- 3.97 (m, 2H); 3.79-3.86 (m, 2H); 3.63-3.92 (m, 1H); 3.47-3.56 (m, 5H); 3.34-3.42 (m, 3H); 1.83-1.90 (m, 2H); 1.52-1.57 (m, 2H); 1.22-1.40 (m, 26H); 1.20 (t, J=7 Hz, 3H); 0.88 (t, J=7 Hz, 3H). MS (EI): 628.40 [M-H]-. HRMS (ESI-) calcd. for C30H55N5O7P [M-H]- 628.3845, found 628.3848 (E = 0.5 ppm). HPLC analysis: retention time 20.92 min., purity 95.1 %.</p><!><p>was synthesized from N4-monomethoxytritylcytosine and (S)-methyl glycidyl ether (procedure A). 91% yield. 1H NMR (CDCl3/methanol-d4), δ 7.45-7.67 (m, 14H); 7.17 (d, J=6Hz, 1H); 5.82 (d, J=6Hz, 1H); 4.30-4.40 (m, 2H); 4.12 (s, 3H); 3.80-3.92 (m, 1H); 3.65-3.75 (m, 2H); 3.65 (s, 3H).</p><!><p>was synthesized from (S)-9-[(3-methoxy-2- hydroxy)propyl]-N4-monomethoxytritylcytosine (38) and octadecyloxyethyl p-toluenesulfonyloxymethylphosphonate (procedure C). 45% yield 1H NMR (CDCl3/methanol-d4), δ 7.10-7.40 (m, 14H); 6.85 (d, J=6Hz, 1H); 5.52 (d, J=6Hz, 1H); 4.20-4.39 (m, 2H); 3.77-4.02 (m, 4H); 3.55-3.65 (m, 1H); 3.43-3.52 (m, 3H); 3.30-3.45 (m, 8H); 1.45-1.65 (m, 2H); 1.18-1.40 (m, 30H); 0.89 (t, J=7 Hz, 3H). MS (ESI): 860.55 [M-H]-.</p><!><p>Octadecyloxyethyl (S)-1-[(3-methoxy-2-phosphonomethoxy)propyl]-N4-monomethoxytritylcytosine (39) (0. 26 g, 0.60 mmol) was added to 80% aq acetic acid and heated to 60 °C for 2 hours. After cooling, the solvent was removed in vacuo and the residue purified by flash column chromatography on silica gel. Elution with 20% MeOH/ CH2Cl2 gave the product (0.1 g, 55%). 1H NMR (CDCl3/methanol-d4) δ 7.80 (d, J = 6 Hz, 1H); 6.00 (d, J = 6 Hz, 1H); 4.04-4.15 (m, 4H); 3.55-3.68 (m, 3H); 3.42-3.53 (m, 2H); 3.35-3.42 (m, 4H); 1.50-1.65 (m, 2H); 1.18-1.38 (m, 30H); 0.88 (t, J = 7 Hz, 3H). MS (ESI+): 590.33 [M+H]+, 612.34 [M+Na]+. HRMS (ESI-) calcd. for C29H55N3O7P [M-H]- 588.3783, found 588.3784 (E = -0.2 ppm). HPLC analysis: retention time 22.75 min., purity 90.9 %.</p><!><p>Reaction of 6-methoxypurine (TCI America, Portland, OR) with (S)-methyl glycidyl ether according to procedure A gave compound 41. 67% yield. 1H NMR (methanol-d4) δ 8.51 (s, 1H, H-8), 8.24 (s, 1H, H-2), 4.74 (dd, 1H, J1′a2′= 3.8 Hz, Jgem=14.2 Hz), 4.28 (dd, 1H, J1′b2′ = 8 Hz, Jgem = 14.2 Hz), 4.18 (s, 3H, Ar-OCH3), 4.14 (m, 1H, H-2′), 3.42 (d, 2H, J3′2′ = 5.2 Hz), 3.37 (s, 3H, -CH2- OCH3).</p><!><p>Reaction of 6-O-methylguanine (Aldrich Chem.) with (S)-methylglycidyl ether (Procedure A) gave compound 42. 78% yield. 1H NMR (methanol-d4) δ 7.81 (s, 1H, H-8); 4.35 (dd, 2H, H-1′); 4.15 (m, 1H, H-2′); 4.05 (s, 3H, Ar-OCH3); 3.39 (d, 2H, H-3′); 3.35 (s, 3H, -OCH3). MS (ESI): m/z 254.08 [M+H]+.</p><!><p>Reaction of compound 41 with hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate according to procedure C afforded compound 43. 29% yield. 1H NMR (methanol-d4) δ 8.51 (s, 1H, H-8); 8.44 (s, 1H, H-2); 4.56 (dd, 1H, H-1′a , J1′a,2′ = 3.6 Hz, Jgem = 14.0 Hz); 4.44 (dd, 1H, H-1′b, J1′b,2′ = 6.6 Hz, Jgem = 14.6 Hz); 4.17 (s, 3H, Ar-OCH3); 3.96 (m, 1H, H-2′); 3.86 (t, 1H, P-O-CHa, J = 6.4 Hz); 3.84 (t, 1H, P-O-CHb, J = 6.4 Hz); 3.78 (dd, 1H, -CHa-P-, JP,CH = 9.2 Hz, Jgem = 12.8 Hz); 3.60 (dd, 1H, - CHb-P-, JP,CH = 9.6 Hz, Jgem = 12.8 Hz); 3.44 (m, 1H, H-2′); 3.43 (t, 2H, -CH2-O-CH2-); 3.36 (t, 2H, -CH2-O-CH2-); 3.32 (s, 3H, -OCH3); 1.76 (pentet, 2H, -O-CH2CH2CH2-O-); 1.49 (m, 2H, - CH2-O-CH2CH2(CH2)13-); 1.28 (m, 26H, -(CH2)13-); 0.89 (t, 3H, -CH3). MS (ESI-) m/z 613.46 [M-H]-. HRMS (ESI-) calcd. for C30H54N4O7P [M-H]- 613.3736, found 613.3739 (E = 0.5 ppm).</p><!><p>Reaction of compound 42 with hexadecyloxypropyl p-toluenesulfonyloxymethylphosphonate according to procedure C afforded compound 44. 34% yield. 1H NMR (methanol-d4) δ 7.95 (s, 1H, H-8), 4.34 (dd, 1H, H-1′a, J1′a2′ = 4 Hz, Jgem = 14.4 Hz); 4.22 (dd, 1H, H-1′b, J1′b2′ = 6.4 Hz, Jgem = 14.4 Hz); 4.04 (s, 3H, Ar-OCH3); 3.91 (m, 1H, H-2′); 3.87 (t, 1H, P-O-CHa-, J = 6.4 Hz); 3.86 (t, 1H, P-O-CHb-, J = 6.4 Hz); 3.75 (dd, 1H, -CHa-P-, JCH,P = 9.4 Hz, Jgem = 13 Hz); 3.64 (dd, 1H, -CHb-P-, JCH,P = 9.2 Hz, Jgem = 12.8 Hz); 3.44 (t, 2H, -CH2-O-); 3.43 (d, 2H, H-3′); 3.35 (t, 2H, -O-CH2); 3.34 (s, 3H, - OCH3); 1.78 (pentet, 2H, -O-CH2CH2CH2-O-); 1.50 (m, 2H, -OCH2CH2(CH2)13-); 1.27 (m, 26H, (CH2)13); 0.89 (t, 3H, -CH3). MS (ESI): m/z 630.35 [M+H]+; 628.37 [M-H]-. HRMS (ESI-) calcd. for C30H55N5O7P [M-H]- 628.3845, found 628.3847 (E = 0.3 ppm). HPLC analysis: retention time 21.60 min., purity 97.0 %.</p><!><p>HCV assays were carried out as previously described using 10,000 FEO replicon cells (BM4-5 or JFH-1 based) per well in 96-well plates.14 Cells and compounds were incubated for 48 to 72 hours with all conditions run in triplicate. Additionally, three to four experimental replicates were completed per compound. Luciferase activity was determined using a micro-plate luminometer (Veritas Microplate Luminometer, Turner Biosystems) according to the manufacturer's instructions (Brightglo, Promega). Relative light units (RLU) for each condition were used to generate a dose response for each compound using Prism (version 4, GraphPad software). The cytotoxicity of each compound was determined using a fluorescent cell viability and death assay (MultiTox-Fluor, Promega). All compounds were tested at concentrations up to 100 μM (0.3% DMSO final concentration).</p><!><p>MT-2 cells (AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health) were maintained in RPMI 1640 supplemented with 10% FBS (JRH Biosciences, Lenexa, Kans.), 10 mM HEPES buffer, 50 IU of penicillin/ml, and 50 μg of streptomycin/ml. HIV-1LAI was obtained from the AIDS Research and Reference Reagent Program. The antiviral activity of each compound was determined by inoculating MT-2 cells with HIV-1LAI at a multiplicity of infection (MOI) of 0.001 TCID50/cell, followed by incubation in the presence of threefold serial drug dilutions (three wells per dilution). Four days after infection, culture supernatants were harvested, lysed with 0.5% Triton X-100, and assayed for p24 antigen concentration using a commercial enzyme-linked immunosorbent assay (ELISA) (Perkin Elmer Life Sciences, Boston, MA). The antiviral activity of each compound is expressed as the EC50, which is the concentration required to inhibit p24 antigen production by 50%. To assess cytotoxicity, MT-2 cells were incubated with drug for 72 hrs and harvested. Flow count beads (Beckman Coulter, Miami, FL) were added to the cell suspension followed by propidium iodide staining and analysis using an Epics Elite flow cytometer (Beckman Coulter). The 50% cytotoxic concentration (CC50) was calculated from the cell counts and viability.17</p>