Emtricitabine Prodrugs with Improved Anti-HIV Activity and CellularUptakeHitesh K. Agarwal,† Bhupender S. Chhikara,† Sitaram Bhavaraju,† Dindyal Mandal,† Gustavo F. Doncel,*,‡

and Keykavous Parang*,†

†Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island 02881, United States‡CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23507, United States

*S Supporting Information

ABSTRACT: Three fatty acyl conjugates of (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC, emtricitabine) weresynthesized and evaluated against HIV-1 cell-free and cell-associated virus and compared with the corresponding parentnucleoside and physical mixtures of FTC and fatty acids.Among all the compounds, the myristoylated conjugate ofFTC (5, EC50 = 0.07−3.7 μM) displayed the highest potency.Compound 5 exhibited 10−24 and 3−13-times higher anti-HIV activity than FTC alone (EC50 = 0.7−88.6 μM) and thecorresponding physical mixtures of FTC and myristic acid (14,EC50 = 0.2−20 μM), respectively. Cellular uptake studies confirmed that compound 5 accumulated intracellularly after 1 h ofincubation and underwent intracellular hydrolysis in CCRF-CEM cells. Alternative studies were conducted using thecarboxyfluorescein conjugated with FTC though β-alanine (12) and 12-aminododecanoic acid (13). Acylation of FTC with along-chain fatty acid in 13 improved its cellular uptake by 8.5−20 fold in comparison to 12 with a short-chain β-alanine.Compound 5 (IC90 = 15.7−16.1 nM) showed 6.6- and 35.2 times higher activity than FTC (IC90 = 103−567 nM) againstmultidrug resistant viruses B-NNRTI and B−K65R, indicating that FTC conjugation with myristic acid generates a more potentanalogue with a better resistance profile than its parent compound.

KEYWORDS: anti-HIV, (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine, cellular uptake, cytotoxicity, fatty acids

■ INTRODUCTION

Emtricitabine [(−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine, FTC,1] is a potent nucleoside reverse transcriptase inhibitor (NRTI)that inhibits human immunodeficiency virus-1 (HIV-1) andhepatitis B virus (HBV).1,2 Thus, FTC is clinically used as ananti-HIV agent in combination with other drugs in highly activeantiretroviral therapy (HAART) and in treatment of HBVinfection.1 FTC was developed as a 5-fluoro derivative of 3TC[lamivudine, (−)-2′,3′-dideoxy-3′-thiacytidine] and displayed4−10 times more potency than 3TC against HIV-1.1−3

Due to structural similarities, FTC and 3TC share a commonmechanism of action and drug resistance patterns.4 FTC isknown to have a better therapeutic index than other similarNRTIs, such as 3TC, 3′-azido-2′,3′-dideoxythymidine (AZT)and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).1,5−7 Asingle point mutation at residue methionine 184 to valine orisoleucine in HIV genome drastically reduces the activity ofFTC against mutant virus (M184 V/I).8−10 Two major reasonsfor the drug resistance are cytidine deamination for both FTCand 3TC and the generation of steric hindrance by isoleucinesubstitution for methionine.8−10 Other mutant viral strains arealso known to decrease the antiviral activity for 3TC and FTCsuch as mutation of methionine 552 to valine and isoleucine(M552 V/I) in HBV.1,11 Some studies suggest that FTC

generates a higher barrier to drug resistance and displays goodsynergism in combination with tenofovir than 3TC.12−14

Furthermore, nucleoside analogues often suffer from poororal bioavailability because of their hydrophilic nature andlimited cellular permeaibility.15 Several nucleoside prodrugshave been designed to enhance cellular uptake of these polarcompounds.15 The polar nature of FTC (calculated Log P forFTC = −1.29) limits its efficient cellular uptake. We havepreviously reported the synthesis and evaluation of fatty acylester prodrugs of 3TC, AZT, d4T, and FLT.5,16−18 Thesestudies showed improvement in the anti-HIV activities ofnucleoside analogues after conjugation with myristic acidanalogues.5,6,16−18 In addition, myristic acid analogues havemoderate activity against N-myristoyltransferase (NMT), acrucial enzyme involved in the life cycle of HIV (e.g., capsidprotein p17, Pr160gag‑pol, Pr55gag, p27nef).19,20 NMT catalyzesthe myristoylation of viral proteins at N-terminal glycine andmakes them more hydrophobic to improve their protein−

Special Issue: Prodrug Design and Target Site Activation

Received: June 29, 2012Revised: July 31, 2012Accepted: August 23, 2012

Article

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protein and protein−membrane interactions.20 Several myristicacid analogues inhibit NMT,21−23 thereby reducing replicationof HIV-1. Our previous studies indicate that the conjugation ofthree fatty acids, myristic acid, 12-azidododecanoic acid, and12-thioethyldodecanoic acid, with NRTIs generates morepotent analogues.5,16−18

Herein, we discuss the synthesis and anti-HIV activity of fattyacyl esters of FTC as nucleoside prodrugs. The selection of thefatty acids, myristic acid, 12-azidododecanoic acid, and 12-thioethyldodecanoic acid, was based on previous results aboutfatty acyl derivatives of other nucleosides.5,16−18 We hypothe-sized that the FTC conjugation with the myristic acid analogueswould enhance the cellular uptake through improved lip-ophilicity. The fatty acyl esters are expected to get hydrolyzedintracellularly to produce two anti-HIV active agents, thenucleoside analogue and the fatty acid targeting RT and NMTenzymes, respectively. Higher uptake into HIV target cells andsustained intracellular release of two active agents would resultin increased antiviral potency and higher barrier to develop-ment of drug resistance. These compounds were envisioned asimproved prodrug nucleosides for microbicide development.Microbicides are topically applied agents designed to prevent orreduce transmission of sexually transmitted infections (STIs),in particular HIV/AIDS.24

■ EXPERIMENTAL SECTIONMaterials and Methods. Emtricitabine (FTC) was

purchased from Euro Asia Trans Continental (Bombay,India). 12-Bromododecanoic acid was purchased from SigmaAldrich Chemical Co. 5(6)-Carboxyfluorescein (FAM) waspurchased from Novabiochem. All the other reagents includingsolvents were purchased from Fisher Scientific. The finalproducts were purified on a PhenomenexGemini 10 μm ODSreversed-phase column (2.1 × 25 cm) with a Hitachi HPLCsystem using a gradient system at a constant flow rate of 17mL/min (Table S1, Supporting Information).The purity of the compounds was confirmed (>95%) by

using a Hitachi analytical HPLC system on a C18 column(Grace Allsphere ODS-2, 3 μm, 150 × 4.6 mm) using agradient system (water:acetonitrile) at constant flow rate of 1mL/min with a UV detection at 265 nm (Table S2, SupportingInformation).The chemical structures of final products were characterized

by nuclear magnetic resonance spectrometry (1H NMR and13C NMR) determined on a Bruker NMR spectrometer (400MHz) and confirmed by a high-resolution PE BiosystemsMariner API time-of-flight electrospray mass spectrometer.Chemical shifts are reported in parts per millions (ppm). Forcellular uptake studies, cells were analyzed by flow cytometry(FACSCalibur: Becton Dickinson) using FITC channel andCellQuest software. Cell-viability studies were conducted usingCellometer Auto T.4 (Nexcelom Biosciences). The real timemicroscopy in live CCRF-CEM cell line with or withoutcompounds was imaged using a ZEISS Axioplan 2 lightmicroscope equipped with transmitted light microscopy with adifferential-interference contrast method and an Achroplan 40×objective.Chemistry. (−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradeca-

noyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (2), (−)-5′-O-(12-Azidododecanoyl)-N4-(4,4′-dimethoxytrityl)-5-fluoro-2′,3′-di-deoxy-3′-thiacytidine (3), and (−)-(4,4′-Dimethoxytrityl)-5′-O-(12-thioethyldodecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thia-cytidine (4). N4-DMTr protected FTC (1) was synthesized

according to the previously reported method.6 Compound 1(250 mg, 0.45 mmol), the corresponding fatty acid (0.90mmol), and HBTU (350 mg, 0.90 mmol) were dissolved in dryDMF (10 mL). DIPEA (2 mL, 15 mmol) was added to thereaction mixture, and stirring was continued overnight at roomtemperature. The reaction mixture was concentrated at reducedpressure, and the residue was purified by reversed phase HPLCusing C18 column and water/acetonitrile as solvents asdescribed above to afford 2−4.

(−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradecanoyl)-5-fluo-ro-2′,3′-dideoxy-3′-thiacytidine (2). HR-MS (ESI-TOF) (m/z): C43H54FN3O6S, calcd, 759.3717; found, 760.3287 [M +H]+, 861.4357 [M+TEA]+, 1520.6604 [2M + H]+.

(−)-5′-O-(12-Azidododecanoyl)-N4-(4,4′-dimethoxytrityl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (3). Yield: 250 mg, 71%.1H NMR (400 MHz, CDCl3, δ ppm): 8.20−9.00 (br s, 1H, 4-NH), 8.07 (d, J = 6.1 Hz, 1H, H-6), 7.22−7.33 (m, 5H, DMTrprotons), 7.17 (d, J = 8.8 Hz, 4H, DMTr protons), 6.83 (d, J =8.8 Hz, 4H, DMTr protons), 6.27−6.31 (br s, 1H, H-1′), 5.34−5.39 (m, 1H, H-4′), 4.65 (dd, J = 12.6 and 3.9 Hz, 1H, H-5″),4.45 (dd, J = 12.6 and 2.6 Hz, 1H, H-5′), 3.71 (s, 6H, DMTr-OCH3), 3.57 (dd, J = 5.1 and 12.6 Hz, 1H, H-2″), 3.20−3.31(m, 3H, CH2N3, H-2′), 2.40 (t, J = 7.3 Hz, 2H, CH2CO),1.55−1.75 (m, 4H, CH2CH2N3, CH2CH2CO), 1.23−1.41 (brm, 14H, methylene protons). 13C NMR (CDCl3, 100 MHz, δppm): 173.12 (COO), 158.62 (C-4), 156.47 (C-2 CO),152.13, 147.33 (DMTr-C), 139.46 (C-5), 129.14, 127.86,127.77 (DMTr-C), 127.09 (C-6), 113.16 (DMTr-C), 87.25 (C-1′), 85.13 (C-4′), 81.44 (DMTr-C-NH), 62.91 (C-5′), 55.27(DMTr-OCH3), 51.49 (CH2N3), 39.16 (C-2′), 33.96, 29.44,29.38, 29.21, 29.14, 29.07, 28.84, 26.71, 24.82 (methylenecarbons). HR-MS (ESI-TOF) (m/z): C41H49FN6O6S, calcd,772.3418; found, 773.9830 [M + H]+.

(−)-(4,4′-Dimethoxytrityl)-5′-O-(12-thioethyldodecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (4). Yield: 240 mg, 70%.1H NMR (400 MHz, CDCl3, δ ppm): 8.50−9.40 (br s, 1H, 4-NH), 8.09 (d, J = 5.8 Hz, 1H, H-6), 7.23−7.34 (m, 5H, DMTrprotons), 7.17 (d, J = 8.8 Hz, 4H, DMTr protons), 6.83 (d, J =8.8 Hz, 4H, DMTr protons), 6.27−6.31 (br s, 1H, H-1′), 5.34−5.38 (br s, 1H, H-4′), 4.66 (dd, J = 12.7 and 3.8 Hz, 1H, H-5″),4.45 (dd, J = 12.7 and 2.1 Hz, 1H, H-5′), 3.79 (s, 6H, DMTr-OCH3), 3.58 (dd, J = 12.1 and 4.6 Hz, 1H, H-2″), 3.23 (d, J =12.1, 1H, H-2′), 2.49−2.58 (m, 4H, CH2SCH2), 2.41 (t, J = 7.4Hz, 2H, CH2CO), 1.52−1.72 (m, 4H, SCH2CH2 ,CH2CH2CO), 1.23−1.43 (br m, 17H, methylene protons).13C NMR (CDCl3, 100 MHz, δ ppm): 173.09 (COO), 158.63(C-4), 156.23 (C2 CO), 151.71, 147.33 (DMTr-C), 139.46(C-5), 129.14, 127.85, 127.77 (DMTr-C), 127.08 (C-6), 113.17(DMTr-C), 87.24 (C-1′), 85.26 (C-4′), 81.44 (DMTr-C-NH),62.84 (C-5′), 55.26 (DMTr-OCH3), 39.23 (C-2′), 33.98, 31.69,29.65, 29.51, 29.41, 29.26, 29.22, 29.18, 28.96, 25.96, 24.82(methylene carbons), 14.84 (CH3). HR-MS (ESI-TOF) (m/z):C43H54FN3O6S2, calcd, 791.3438; found, 792.5628 [M + H]+.

(−)-5′-O-(Tetradecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacy-tidine (5), (−)-5′-O-(12-Azidododecanoyl)-5-fluoro-2′,3′-di-deoxy-3′-thiacytidine (6), and (−)-5-Fluoro-5′-O-(12-thioe-thyldodecanoyl)-2′,3′-dideoxy-3′-thiacytidine (7). Acetic acid(80%, 10 mL) was added to compounds 2−4 (0.3 mmol). Thereaction mixture was heated at 80 °C for 30 min. The reactionmixture was concentrated at reduced pressure, and the residuewas purified by reversed phase HPLC using C18 column andwater/acetonitrile as solvents as described above to yield 5−7.

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(−)-5′-O-(Tetradecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacy-tidine (5). Yield: 120 mg, 87%. 1H NMR (400 MHz, CDCl3, δppm): 8.03 (d, J = 6.2 Hz, 1H, H-6), 6.25−6.29 (m, 1H, H-1′),5.33−5.37 (m, 1H, H-4′), 4.63 (dd, J = 12.6 and 4.1 Hz, 1H, H-5″), 4.43 (dd, J = 12.6 and 2.2 Hz, 1H, H-5′), 3.56 (dd, J = 12.6and 5.2 Hz, 1H, H-2″), 3.23 (d, J = 12.6 Hz, 1H, H-2′), 2.29−2.43 (m, 2H, CH2CO), 1.55−1.73 (m, 2H, CH2CH2CO),1.15−1.49 (br m, 20H, methylene protons), 0.89 (s, J = 6.2 Hz,3H, CH3).

13C NMR (CDCl3, 100 MHz, δ ppm): 173.15(COO), 157.20 (J = 16.4 Hz, C-4), 152.07 (C-2 CO),135.94 (J = 240.1 Hz, C-5), 126.29 (J = 32.4 Hz, C-6), 87.26(C-1′), 85.02 (C-4′), 63.00 (C-5′), 39.00 (C-2′), 34.22(CH2CO), 31.93, 29.65, 29.61, 29.46, 29.36, 29.28, 29.24,29.11, 24.83, 22.70 (methylene carbons), 14.13 (CH3). HR-MS(ESI-TOF) (m/z): C22H36FN3O4S, calcd, 457.2411; found,458.0814 [M + H]+, 915.1334 [2M + H]+.(−)-5′-O-(12-Azidododecanoyl)-5-fluoro-2′,3′-dideoxy-3′-

thiacytidine (6). Yield: 125 mg, 88%. 1H NMR (400 MHz,CDCl3, δ ppm): 8.90−9.70 (br s, 2H, 4-NH2), 8.05 (d, J = 5.9Hz, 1H, H-6), 6.25−6.29 (m, 1H, H-1′), 5.33−5.37 (m, 1H, H-4′), 4.62 (dd, J = 12.6 and 4.0 Hz, 1H, H-5″), 4.43 (dd, J = 12.6and 1.8 Hz, 1H, H-5′), 3.56 (dd, J = 12.6 and 5.2 Hz, 1H, H-2″), 3.24 (t, J = 6.7 Hz, 3H, H-2′, CH2N3), 2.30−2.43 (m, 2H,CH2CO), 1.53−1.69 (m, 4H, CH2CH2CO, CH2CH2N3),1.20−1.40 (br m, 14H, methylene protons). 13C NMR(CDCl3, 100 MHz, δ ppm): 173.13 (COO), 156.96 (J = 16.0Hz, C-4), 151.89 (C-2 CO), 135.95 (J = 237.1 Hz, C-5),126.45 (J = 32.7 Hz, C-6), 87.25 (C-1′), 85.05 (C-4′), 63.00(C-5′), 51.47 (CH2N3), 38.90 (C-2′), 34.21 (CH2CO), 29.42,29.36, 29.23, 29.18, 29.11, 29.08, 29.04, 28.82, 26.69, 24.79,24.76 (methylene carbons). HR-MS (ESI-TOF) (m/z):C20H31FN6O4S, calcd, 470.2112; found, 471.0575 [M + H]+,941.0986 [2M + H]+.(−)-5-Fluoro-5′-O-(12-thioethyldodecanoyl)-2′,3′-di-

deoxy-3′-thiacytidine (7). Yield: 110 mg, 80%. 1H NMR (400MHz, CDCl3, δ): 8.07 (d, J = 6.1 Hz, 1H, H-6), 6.26−6.30 (m,1H, H-1′), 5.37 (t, J = 2.4 Hz, 1H, H-4′), 4.65 (dd, J = 12.6 and4.1 Hz, 1H, H-5″), 4.45 (dd, J = 12.6 and 2.4 Hz, 1H, H-5′),3.58 (dd, J = 12.7 and 5.3 Hz, 1H, H-2″), 3.23 (dd, J = 12.7 and2.1 Hz, 1H, H-2′), 2.49−2.58 (m, 4H, CH2SCH2), 2.30−2.45(m, 2H, CH2COO), 1.53−1.69 (m, 4H, SCH2CH2,CH2CH2CO), 1.24−1.42 (br m, 17H, methylene protons).13C NMR (CDCl3, 100 MHz, δ ppm): 173.08 (COO), 156.00(J = 17.2 Hz, C-4), 150.87 (C-2 CO), 135.67 (J = 239.3 Hz,C-5), 126.79 (J = 32.5 Hz, C-6), 87.20 (C-1′), 85.46 (C-4′),62.75 (C-5′), 39.21 (C-2′), 33.96 (CH2COO), 31.68, 29.65,29.50, 29.40, 29.32, 29.24, 29.15, 29.07, 28.96, 28.88, 25.92,24.80, 24.73 (methylene carbons), 14.83 (CH3). HR-MS (ESI-TOF) (m/z): C22H36FN3O4S2, calcd, 489.2131; found,490.4833 [M + H]+.(−)-5-Fluoro-5′-O-(3(N-Fmoc-aminopropanoyl)-N4-(4,4′-

dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (8) and (−)-5-Fluoro-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dime-thoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (9). Compound 1(320 mg, 0.60 mmol), the corresponding Fmoc-amino acid(1.2 mmoL), and HBTU (500 mg, 1.3 mmol) were dissolved ina mixture of dry DMF (10 mL) and DIPEA (2 mL, 15 mmol).The reaction mixture was stirred overnight at room temper-ature. The reaction mixture was concentrated and dried underreduced pressure to afford crude 5′-O-Fmoc-amino acidderivatives of N4-DMTr-2′,3′-dideoxy-3′-thiacytidine, 8 and 9.(−)-5-Fluoro-5′-O-(3(N-Fmoc-aminopropanoyl)-N4-(4,4′-

dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (8). HR-MS

(ESI-TOF) (m/z): C47H43FN4O8S, calcd, 842.2786; found,843.2138 [M + H]+.

(−)-5-Fluoro-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (9). HR-MS (ESI-TOF) (m/z): C56H61FN4O8S, calcd, 968.4194; found,991.4431 [M + Na]+.

(−)-5-Fluoro-5′-O-(3-aminopropanoyl)-N4-(4,4′-dimethox-ytrityl)-2′,3′-dideoxy-3′-thiacytidine (10) and (−)-5-Fluoro-5′-O-(12-aminododecanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (11). The crude products weredissolved in piperidine (20% in DMF, 10 mL), and thereaction mixture was stirred for 1 h at room temperature. Thereaction solution was concentrated at reduced pressure. Theresidue was purified with reversed phase HPLC using C18column and water/acetonitrile as solvents as described above toyield 10 and 11.

(−)-5-Fluoro-5′-O-(3-aminopropanoyl)-N4-(4,4′-dimethox-ytrityl)-2′,3′-dideoxy-3′-thiacytidine (10). Overall yield: 200mg, 55%. HR-MS (ESI-TOF) (m/z): C32H33FN4O6S, calcd,620.2105; found, 621.2401 [M + H]+.

(−)-5-Fluoro-5′-O-(12-aminododecanoyl)-N4-(4,4′-dime-thoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (11). Overall yield:210 mg, 52%. 1H NMR (400 MHz, CDCl3, δ ppm): 7.79 (d, J= 6.8 Hz, 1H, H-6), 7.20−7.35 (m, 5H, DMTr-H), 7.17 (d, J =8.8 Hz, 4H, DMTr-H), 6.81 (d, J = 8.8 Hz, 4H, DMTr-H),6.20−6.30 (m, 1H, H-1′), 6.00 (br s, 1H, N4-NH), 5.32−5.36(m, 1H, H-4′), 4.54 (dd, J = 12.4 and 4.8 Hz, 1H, H-5″), 4.40(dd, J = 12.4 and 2.9 Hz, 1H, H-5′), 3.78 (s, 6H, DMTr-CH3),3.54 (dd, J = 12.2 and 5.4 Hz, 1H, H-2″), 3.07 (dd, J = 3.6 and12.2 Hz, 1H, H-2′), 2.80 (t, J = 7.6 Hz, 2H, CH2NH2), 2.38 (t,J = 7.4 Hz, 2H, CH2CO), 1.55−1.75 (m, 4H, CH2CH2NH andCH2CH2CO), 1.43 (t, J = 7.2 Hz, 2H, CH2NH2), 1.20−1.40(br m, 14H, methylene protons). 13C NMR (CDCl3, 100 MHz,δ ppm): 173.88 (COO), 161.67 and 161.34 (DMTr-C), 158.03(J = 16.0 Hz, C-4), 153.13 (C-2 CO), 137.19 (J = 239.2 Hz,C-5), 127.21 (J = 33.0 Hz, C-6), 122.29, 119.36, 116.14, 113.52(DMTr-C), 88.14 (C-1′), 85.39 (C-4′), 64.05 (C-5′), 59.20(DMTr-OCH3), 51.84 (CH2NH2), 40.43 (C-2′), 38.75(CH2CO), 34.87 (CH2CH2NH2), 30.15, 30.14, 30.04, 29.96,29.82, 29.73, 29.59, 28.01, 27.02, 25.58 (methylene carbons).HR-MS (ESI-TOF) (m/z): C41H51FN4O6S, calcd, 746.3513;found, 747.4272 [M + H]+.

General Procedure for the Synthesis of 5′-O-(5(6)-Carboxyfluorescein) Derivatives of FTC (12 and 13). Amixture of 5(6)-carboxyfluorescein (430 mg, 1.15 mmol), thecorresponding N4-DMTr-5′-O-aminoacyl derivative of FTC(10 or 11, 0.29 mmoL), and HBTU (440 mg, 1.15 mmol) wasdissolved in a mixture of dry DMF (10 mL) and DIPEA (2 mL,15 mmol) and stirred overnight at room temperature. Thereaction mixture was concentrated and dried under vacuum.Acetic acid (80%, 10 mL) was added to the reaction mixtureand was heated at 80 °C for 30 min. The final compounds (12and 13) were purified with reversed phase HPLC using C18column and using water/acetonitrile as solvents as describedabove.

(−)-5-Fluoro-5′-O-(3-(N(5(6)-carboxyfluorescein)-aminopropanoyl)-2′,3′-dideoxy-3′-thiacytidine (12). Yield:40 mg, 20%. 1H NMR (400 MHz, CD3OD, δ ppm): 8.45 (s,0.70 H, FAM-Ar-H, 5 or 6 isomer), 8.31 (d, J = 6.7 Hz, 0.8 H,H-6), 8.19 (dd, J = 1.6 and 8.1 Hz, 1H, FAM-Ar-H, 5 or 6isomer), 8.11 (s, 0.5 H, FAM-Ar-H, 5 or 6 isomer), 7.16−7.40(m, 2 H, FAM-Ar-H, 5 or 6 isomer), 6.60−6.80 (m, 4H, FAM-Ar-H), 6.08−6.14 and 6.14−6.23 (two m, 1H, H-1′), 5.47 and

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5.36 (dd, J = 2.8 and 4.4 Hz, 1 H, H-4′), 4.77 and 4.64 (two dd,J = 4.5 and 12.6 Hz, 1H, H-5″), 4.37 and 4.49 (dd, J = 2.7 and12.6 Hz, 1H, H-5′), 3.68−3.82 (m, 2 H, CH2NH), 3.13−3.21(m, 1H, H-2″ and H-2′), 2.65−2.85 (m, 2H, CH2CO).

13CNMR (CD3OD, 100 MHz, δ ppm): 172.84 (COO), 169.96(CONH), 168.27 (COO-FAM), 163.48, 163.52 (Ar-C-FAM),156.88 (J = 20.9 Hz, C-4), 156.25, 153.23 (Ar-C-FAM), 150.93(C-2 CO), 141.27, 137.72 (Ar-C-FAM), 137.08 (J = 238.5Hz, C-5), 134.99, 131.01, 130.92, 129.33, 129.06, 128.72 (Ar-C-FAM), 126.41 (J = 32.3 Hz, C-6), 121.50, 115.14, 112.07 and112.04, (Ar-C-FAM), 103.61 (C-5), 88.97 (C-1′), 86.35 (C-4′),64.71 (C-5′), 38.88 (C-2′), 37.24 (CH2CONH), 34.80(CH2COO). HR-MS (ESI-TOF) (m/z): C32H25FN4O10S,calcd, 676.1275; found, 677.1633 [M + H]+.(−)-5-Fluoro-5′-O-(12-(N(5(6)-carboxyfluorescein)-

aminododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (13). Yield:30 mg, 16%. 1H NMR (400 MHz, CD3OD, δ ppm): 8.81−8.93(s, 0.37 H, FAM-Ar-H, 5 or 6 isomer), 8.62 (t, J = 7.8 Hz, 0.17H, FAM-Ar-H, 5 or 6 isomer) 8.49 (s, 0.63 H, FAM-Ar-H, 5 or6 isomer), 8.28 (m, 1 H, FAM-Ar-H, 5 or 6 isomer), 8.20 (d, J= 8.2 Hz, 0.8 H, FAM-Ar-H, 5 or 6 isomer), 8.09 (dd, J = 11.7and 5.0 Hz, 1H, H-6), 7.01 (s, 0.30 H, FAM-Ar-H), 7.31 (d, J =7.8 Hz, 0.5H, FAM-Ar-H, 5 or 6 isomer), 6.66−6.82 (m, 4H,FAM-Ar-H), 6.55−6.66 (m, 2H, FAM-Ar-H), 6.12−6.25 (m,1H, H-1′), 5.33−5.43 (m, 1H, H-4′), 4.64 (dd, J = 12.5 and 4.0Hz, 1H, H-5″), 4.40 (d, J = 12.5 Hz, 1H, H-5′), 3.54 (dd, J =5.5 and 12.5 Hz, 1H, H-2″), 3.41 (t, J = 6.6 Hz, 2H, CH2NH),3.20−3.39 (m, 1H, H-2′), 2.27−2.41 (m, 2H, CH2COO),1.45−1.70 (m, 4H, CH2CH2CO and CH2CH2NH), 1.10−1.40(br m, 16H, methylene protons). 13C NMR (CD3OD, 100MHz, δ ppm): 172.08 (COO), 171.41 (CONH), 166.72(COO-FAM), 164.91, 164.78, 160.43 (Ar-C-FAM), 153.61 (J =20.6 Hz, C-4), 152.08 (Ar-C-FAM), 149.54 (J = 4.2 Hz, C-2CO), 147.92, 144.87, 139.83, 138.76, 134.87 (Ar-C-FAM),133.83 (J = 238.1 Hz, C-5), 131.74 (Ar-C-FAM), 127.83,126.11 (Ar-C-FAM), 125.76 (J = 32.3 Hz, C-6), 125.52, 123.68,123.08, 112.12, 109.01, (Ar-C-FAM), 100.52 (C-5), 85.52 (C-1′), 83.34 (C-4′), 60.99 (C-5′), 38.19 (C-2′), 35.98(CH2COO), 31.65, 27.50, 27.44, 27.40, 27.34, 27.26, 27.17,27.12, 26.96 (methylene carbons), 24.93 (CH2CH2NH2), 22.79(CH2CH2COO). HR-MS (ESI-TOF) (m/z): C41H43FN4O10S,calcd,802.2684; found, 803.3122 [M + H]+.Anti-HIV Assays. The anti-HIV activity of the compounds

was evaluated according to previously reported methods.5,25−27

Compound anti-HIV activity was evaluated in single-round(MAGI) infection assays using X4 (IIIB) and R5 (BaL) HIV-1and P4R5 cells expressing CD4 and coreceptors. Briefly,P4R5MAGI cells were cultured at a density of 1.2 × 104 cells/well in a 96 well plate approximately 18 h prior to infection.Cells were incubated for 2 h at 37 °C with purified, cell-freeHIV-1 laboratory strains IIIB or BaL (Advanced Biotechnol-ogies, Inc., Columbia, MD) in the absence or presence of eachagent. After 2 h, cells were washed, cultured for an additional 46h, and subsequently assayed for HIV-1 infection using theGalacto-Star β-Galactosidase Reporter Gene Assay System forMammalian Cells (Applied Biosystems, Bedford, MA).Reductions in infection were calculated as a percentage relativeto the level of infection in the absence of agents, and 50%inhibitory concentrations (EC50) were derived from regressionanalysis. Each compound concentration was tested in triplicatewells. Cell toxicity was evaluated using the same experimentaldesign but without the addition of virus. The impact ofcompounds on cell viability was assessed using an MTT

(reduction of tetrazolium salts) assay (Invitrogen, Carlsbad,CA).For the assessment of compounds against wild-type (WT;

R5; clones = 94US3393IN [B subtype] and 98USMSC5016 [Csubtype]) and drug resistant (clones = 4755-5, 71361-1, andA17) HIV-1 clinical isolates, phytohemagglutinin stimulatedperipheral blood mononuclear cells (PBMCs) from at least twonormal donors were pooled, diluted in fresh media, and platedin the interior wells of a 96 well round-bottom microplate. Eachplate contained virus/cell control wells (cells + virus),experimental wells (drug + cells + virus), and compoundcontrol wells (drug + media, no cells, necessary for MTSmonitoring of cytotoxicity). Test drug dilutions were preparedin microtiter tubes, and each concentration was placed inappropriate wells. Following addition of the drug dilutions tothe PBMCs, a predetermined dilution of virus stock was thenplaced in each test well (final MOI ≅ 0.1). Since HIV-1 is notcytopathic to PBMCs, the same assay plate can be used for bothantiviral efficacy and cytotoxicity measurements. Compoundswere incubated with the virus and cells in a 96 well format for 6h. The cells were then washed by removing 75% of the medium(150 μL) and replacing with 150 μL of fresh (no drug)medium. The plates were then centrifuged (∼200g) for 10 min,after which 150 μL of medium was removed and an additional150 μL of fresh medium was added to each well and furtherincubated for 6 days or until peak reverse transcriptase (RT)activity was detected. A microtiter plate-based RT reaction wasutilized.28 Incorporated radioactivity (counts per minute,CPM) was quantified using standard liquid scintillationtechniques. Compound IC50 (50%, inhibition of virusreplication) was calculated using statistical software andregression analysis.

Cellular Uptake Studies. All of the stock solutions forcompounds FAM, 5, 12, and 13 were prepared in DMSO. Thehuman T lymphoblastoid cells CCRF-CEM (ATCC No. CCL-119) were grown on 25 cm2 cell culture flasks with RMPI-1640medium containing 10% fetal bovine serum. Upon reachingabout 70% confluency, the cells were treated as described belowand incubated for 1−24 h or longer at 37 °C.

Cellular Uptake of 5 at Different Time Points. Cellularuptake and accumulation of compound 5 was evaluated inCCRF-CEM cells by analytical HPLC studies. CCRF-CEMcells were grown in 75 cm2 culture flasks with serum free RPMImedium to ∼70−80% confluency (∼1 × 106 cells/mL). Themedium was replaced with RPMI medium containingcompound 5 (50 μM). The cells were incubated at 37 °C for1 h, 12 h, and 24 h. After incubation for the indicated time, thecells were collected by centrifugation. The medium wasremoved carefully by decantation, and the cell pellets werewashed with ice-cold PBS to remove any medium. The cellpellets were thoroughly extracted with an equal volume ofmethanol, chloroform, and isopropanol mixture (100 μL, 4:3:1v/v/v) and filtered through 0.2 μm filters. Compound 5 in celllysates was detected by analytical HPLC analysis (20 μLinjection) at 265 nm using a gradient of water (0.1% TFA)/acetonitrile (0.1% TFA) and C18 column chromatography(Table S2, Supporting Information). The retention time forcompound 5 was 18.2 min with this gradient system. The areaunder the curve for 5 after 1 h, 12 h, and 24 h was normalizedper million cells, and the values (AUC/million cells) werecompared.

Cellular Uptake Study of Fluorescence-Labeled NucleosideAnalogues (12 and 13). All of the stock solutions for

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compounds FAM, 12, and 13 were prepared in DMSO. Thehuman T lymphoblastoid cells CCRF-CEM (ATCC No. CCL-119) were grown on 25 cm2 cell culture flasks with RMPI-1640medium containing 10% fetal bovine serum. When the cellsreached about 70% confluency, FAM, 12, or 13 (1 mL, 20 μM)in RMPI-1640 medium was added to 1 mL of cells to make thefinal concentration 10 μM. The cells were incubated for 1 h at37 °C. After 1 h of incubation, the cells were collected bycentrifugation at 2000 rpm for 5 min. The cells were thenwashed with PBS (pH 7.4) three times. They were resuspended

in flow cytometry buffer, and the flow cytometry assays wereperformed as described below.

Cellular Uptake of 12 and 13 with Trypsin Treatment.When the cells reached about 70% confluency, FAM, 12, or 13(1 mL, 10 μM) in RMPI-1640 medium was added to 1 mL ofcells to make the final concentration 10 μM. The cells wereincubated for 1 h at 37 °C. Then the cells were treated with0.25% trypsin/0.53 mM EDTA at room temperature for 5 minto remove any artificial uptake on the cell surface followed bywashing with PBS (pH 7.4) three times. Washed cells were

Scheme 1. Synthesis of Fatty Acyl Ester Derivatives of FTC

Table 1. Comparison of Anti-HIV Activities of Fatty Acyl Derivatives of FTC with Physical Mixtures of FTC + Fatty Acidsa

antiviral activityc

compound cell-free HIV-1 cell-associated HIV-1

code name cytotoxicity:b CC50 (μM) X4d EC50 (μM) R5e EC50 (μM) X4f EC50 (μM) Log P (calcd)g

1 FTC >200 1.9 0.7 88.6 −1.295 5′-O-myristoyl FTC >200 0.1 0.07 3.7 5.966 5′-O-(12-azidododecanoyl) FTC >200 0.8 0.2 9.1 5.177 5′-O-(12-thioethyldodecanoyl) FTC 93.6 0.05 0.04 4.9 5.0514 myristic acid + FTC (50:50) >200 1.3 0.2 20 NDh

15 12-thioethyldodecanoic acid + FTC (50:50) >200 0.2 0.4 19.3 NDDMSO dimethyl sulfoxide >1000 >1000 >1000 >1000 NDC-2i dextran sulfate >25 0.02 0.4 0.1 ND

aData are expressed as 50% effective concentration (CC50 for cytotoxicity and EC50 for antiviral activity).bCytotoxicity assay (MTS). cSingle-round

infection assay. dLymphocytotropic strain, IIIb. eMonocytotropic strain, BaL. fCell-associated transmission assay (IIIB). gCalculated partitioncoefficient using ChemBioDraw Ultra 12.0. hNot determined. iPositive assay control (dextran sulfate (50 KDa).

Scheme 2. Synthesis of 5′-Carboxyfluorescein Conjugates of FTC (12 and 13)

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resuspended in flow cytometry buffer and the flow cytometryassays were performed as described below.Flow Cytometry. The cells were analyzed by flow cytometry

(FACS Calibur: Becton Dickinson) using FITC channel andCellQuest software. The data presented are based on the meanfluorescence signal for 10000 cells collected. All the assays weredone in triplicate.Real Time Fluorescence Microscopy in the Live CCRF-CEM

Cell Line. The cellular uptake studies and intracellularlocalization of CCRE-CEM cells alone or incubated with 12and 13 were imaged using a ZEISS Axioplan 2 light microscopeequipped with transmitted light microscopy with a differential-interference contrast method and an Achroplan 40× objective.The human T lymphoblastoid cells CCRF-CEM (ATCC No.CCL-119) were grown on 60 mm Petri dishes with RPMI-1640medium containing 10% fetal bovine serum. Upon reachingabout 70% confluency, the cells were incubated with a solutionof 10 μM of 12 and 13 for 1 h at 37 °C. They were then

observed under the fluorescent microscope under bright fieldand FITC channels (480/520 nm).

■ RESULTS AND DISCUSSIONChemistry. 5′-O-Fatty Acyl Ester Derivatives of FTC. 5′-O-

Fatty acyl esters of FTC (5−7) were synthesized (Scheme 1)

Table 2. Anti-HIV Activity of 5′-O-Fatty Acyl Derivatives of FTC against Clinical Isolates and Drug Resistant Viruses

compd virus clade/resistance IC50a (nM) IC90

b (nM) CC50c (nM) TId

FTC 94US3393IN B 12.1 32.4 >12144 >100498USMSC5016 C 2.8 161.9 >12144 >4337A-17 MDR B-NNRTI 35.1 102.8 >12144 34671361-1 MDR B-K65R 16.0 566.7 >12144 >7594755-5 MDR B-MDR 5966.6 >12144 >12144 >2

5 94US3393IN B <0.7 6.6 >6561 >937398USMSC5016 C 1.1 10.9 >6561 >5965A-17 MDR B-NNRTI 1.6 15.7 >6561 410171361-1 MDR B-K65R 1.5 16.1 >6561 >43744755-5 MDR B-MDR 1747.4 >6561 >6561 >3.8

aIC50: 50% inhibitory concentration. bIC90: 90% inhibitory concentration. cTC50: 50% cytotoxic concentration. dTI: Therapeutic index. Multipleround of infection; PBMC-based assay (p24 end point).

Figure 1. HPLC chromatograms for the cellular uptake studies of 5 using CCRF-CEM cells: (A) blank, (B) incubation for 1 h, (C) incubation for 12h, and (D) incubation for 24 h (x axis = minutes and y axis = mAU).

Figure 2. Cellular uptake studies for 12 and 13 with DMSO and FAMas controls with and without treatment with trypsin.

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by the conjugation of fatty acids, myristic acid, 12-azidododecanoic acid, and 12-thioethyldodecanoic acid withFTC. 5′-O-Substitution and selection of fatty acids were basedon our recently published work on the conjugation of similarfatty acids with 3TC.16

5′-O-Fatty acyl derivatives of FTC (5−7) were synthesizedfrom N4-DMTr protected FTC (1), which was synthesizedaccording to the previously reported method.6 Compound 1was reacted with the fatty acids (Scheme 1) in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluor-ophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA)in dimethylformamide at room temperature, overnight, toproduce (2−4). Compounds 2−4 were dissolved in acetic acid(80% in water) and heated at 80 °C for 0.5 h to generate 5′-O-(fatty acyl) esters of FTC (5−7). These compounds werepurified by semipreparative HPLC. The purity of final products(>95%) was confirmed by analytical HPLC (Table S1,Supporting Information). The conjugation of the long chainfatty acids to FTC increased their partition coefficients (Log P)(Table 1), resulting in enhanced lipophilicity in comparisonwith the parent nucleoside analogue.5(6)-Carboxyfluorescein Conjugates of FTC. 5(6)-Carboxy-

fluorescein (FAM) was conjugated with FTC using twodifferent spacers, β-alanine and 12-aminododecanoic acid(Scheme 2). Initially, FTC-DMTr (1) was reacted with thecorresponding N-Fmoc-amino acids in the presence of HBTUand DIPEA to produce DMTr-protected FTC conjugates of the

corresponding N-Fmoc protected amino acid amides of FTC(8 and 9). The N-Fmoc groups were deprotected using crudereaction mixtures of 8 and 9 in the presence of piperidine toproduce amino acid amides of FTC (10 and 11). Finally, FAMwas attached to the free amino group in the presence of HBTUand DIPEA, followed by N4-DMTr deprotection in thepresence of acetic acid (80% in water) to afford the 5(6)-carboxyfluorescein derivatives of FTC (12 and 13, Scheme 2).These compounds were used to determine the cellular

uptake profile of the fatty acyl esters of FTC. FTC conjugatecarboxyfluorescein through β-alanine (12) was used as acontrol FTC analogue whereas FTC attached to FAM through12-aminododecanoyl (13) was used as an analogue of (−)-5-fluoro-5′-O-(12-azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine(6) and other 5′-O-fatty acyl ester derivatives of FTC.

Biological Activities. Fatty acyl conjugates of FTC (5−7)were tested for their cytotoxicity and antiviral activity againstcell-free, cell-associated, and multidrug resistant HIV-1. Theantiviral activity of the fatty acyl ester derivatives of FTC isdescribed in Table 1, and the results are compared with theparent nucleoside FTC alone and its physical mixtures with thecorresponding fatty acids. The synthesized conjugates 5 and 6,and FTC, displayed no cytotoxicity at the highest testedconcentrations (CC50 > 200 μM), whereas 7 showed a CC50 =93.6 μM.5′-O-(Fatty acyl) esters of FTC (5−7) were consistently

active against both cell-free and cell-associated virus, and

Figure 3. Real time fluorescence microscopy in live CCRF-CEM cell line for control (DMSO) and 5(6)-carboxyfluorescein derivatives of FTC (12and 13).

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exhibited higher anti-HIV activity than FTC (Table 1). TheFTC esters displayed the highest anti-HIV activity against cell-free virus (EC50 = 0.04−0.2 μM) among all the previouslyreported fatty acyl ester derivatives and their parentNRTIs.5,16−18

Myristic acid conjugate of FTC (5) showed high anti-HIVactivity against cell-free virus (EC50 = 0.07−0.1 μM), which was∼10−19 times better than that of FTC alone (EC50 = 0.7−1.9μM). In addition, the anti-HIV activity against cell-associatedvirus for 5 (EC50 = 3.7 μM) was ∼24 times higher than that ofFTC (EC50 = 88.6 μM). The 5′-O-12-thioethyldodecanoylderivative of FTC (7) displayed slightly higher anti-HIV activitythan 5, but showed higher cytotoxicity (CC50 = 93.6 μM).In order to compare the efficiency of the conjugation, the

anti-HIV activities of the conjugates 5 and 7 were furthercompared with their corresponding physical mixtures. Theequimolar (50:50) physical mixture of FTC with myristic acid(14) and 12-thioethyldodecanoic acid (15) showed signifi-cantly less potency (EC50 = 0.2−20 μM) than thecorresponding 5′-O-fatty acyl ester derivatives, 5 (EC50 =0.07−3.7 μM) and 7 (EC50 = 0.04−4.9 μM), respectively.Compound 5 showed the best increase in activity, approx-imately 3−13 times higher active than the correspondingphysical mixture 14 (EC50 = 0.2−20 μM) against both cell-freeand cell-associated virus (Table 1). These data suggest that thecellular uptake of the conjugates and intracellular hydrolysis toparent analogue and fatty acid contribute significantly to thehigher anti-HIV profile of the ester conjugates versus thephysical mixtures, and therefore, it is critically important to theimprovement of viral inhibition. The anti-HIV activity of thephysical mixtures was slightly higher than that of FTC.Comparative evaluation of FTC with the 14 and 15 indicatedthat the physical mixing produced a cooperative/additive anti-HIV effect possibly through targeting two independentenzymes used in the HIV life cycle.Compound 5 was selected for further testing against clinical

isolates of HIV, and the results were compared with those ofFTC (Table 2). The results indicated that myristoylconjugation with FTC (5) improved the anti-HIV activity ofFTC against wild-type and drug resistant virus by several-fold.The anti-HIV activity of 5 against clade B and C clinical isolates(IC90 = 6.6 and 10.9 nM, respectively) was approximately 5-and 15-fold higher than that of FTC (IC90 = 32.4 and 161.9nM). When tested against drug resistant viruses bearingmutations that confer resistance to nonnucleoside reversetranscriptase inhibitors (B-NNRTI) and the lead NRTItenofovir (B-K65R), the IC90 values of 5 were 15.7 nM and16.1 nM, respectively, being 6.6 and 35.2 times lower thanthose of FTC (IC90 = 103 and 567 nM). Analogue 5 was betterthan FTC even when compared to the multidrug resistant virus4755-5, which confers resistance to numerous RTIs. The lowerIC50 and IC90 values for 5 in comparison to FTC increased itstherapeutic index. These results suggest that fatty acyl esterconjugate 5 has improved antiviral potency against wild-typeand drug resistant HIV-1 clinical isolates.Cellular Uptake Studies. As shown by calculated Log P

values (Table 1), the presence of 5′-O-substituted long chainfatty acids enhances the lipophilicity of the nucleosidederivatives. Previous studies on fatty acyl conjugates of d4Tand 3TC indicated that fatty acyl conjugation resulted inimproved lipophilicity and cellular uptake of nucleosideconjugates. Therefore, 5′-O-fatty acyl conjugates of FTC werealso expected to have improved cellular uptake in comparison

to FTC alone. In order to better understand the cellular uptakeprofile of the FTC conjugates, the studies were conducted for 5using human T lymphoblastoid cells (CCRF-CEM, ATCC No.CCL-119). Cells were grown to 70% confluency in the culturemedia and then incubated with 5 (50 μM) for 1−24 h at 37 °C.After incubation, cells were centrifuged at the indicated time,extracted, and analyzed by HPLC using C18 columnchromatography and detection at 265 nm (see Materials andMethods for more details).HPLC analysis (Figure 1A) confirmed cellular uptake of

conjugate 5 after 1 h of incubation (retention time: 18.2 min).Cell extracts for samples after 12 h incubation showed asignificant decrease in the intracellular levels of 5. The resultssuggest that the conjugate was hydrolyzed intracellularly toFTC in the presence of intracellular esterases over the period oftime. FTC and peaks of potentially phosphorylated productswere observed at 1.7−2.9 min in the HPLC profile, suggestingintracellular hydrolysis. Conjugate 5 disappeared completely inthe cellular extracts after 24 h. A strong peak was detected at1.7−2.9 min, indicating the presence of metabolic products of5. It is possible that FTC and its phosphorylated formsoverlapped with cell extract peaks. These results suggest thatconjugate 5 was able to cross the cell membrane and washydrolyzed intracellularly as required for a prodrug to becomeactive. However, overlapping peaks for FTC and thephosphorylated products with other intracellular cellularcontents make this method inappropriate for comparativestudies with the parent analogue. Thus, fluorescence-labeledfatty acyl analogues of FTC were synthesized for comparativestudies.For a direct comparison between FTC and the FTC

conjugates, cellular uptake studies were performed usingFAM conjugates of FTC. FTC was attached to FAM throughβ-alanine (12) and 12-aminododecanoic acid (13) as theanalogues of FTC and 6, respectively. CCRF-CEM cells weregrown to 70% confluency in the culture media before they wereincubated with the fluorescein-substituted conjugates, 12 and13 (10 μM), for 1 h either in the presence or in the absence oftrypsin (Figure 2). DMSO and FAM were used as controls forthe study. Incubated cells were analyzed by flow cytometry(FACSCalibur: Becton Dickinson) using FITC channel andCellQuest software. The data are presented as the meanfluorescence signal for 10000 cells collected. The results clearlyindicated that the presence of a long chain spacer betweenFAM and FTC improved the cellular uptake. Cellular uptake of13 was 20-fold higher than that of 12. To confirm that theenhanced uptake of 13 was not just due to the outer cellmembrane adherence, half of the cells from each batch weretreated with trypsin for 5 min to wash the adsorbed moleculesfrom the plasma cell membrane. The cellular uptake of 13 fortrypsin treated cells was 8.5-fold higher than that of 12. Resultssuggest that a great proportion of cellular uptake of 13 wasgoverned by improved lipophilicity and was not due toadsorption to the cell membrane.

Real Time Fluorescence Microscopy in Live CCRF-CEMCells. CCRF-CEM cells were incubated with DMSO, 12, and13 (10 μM) for 1 h and imaged using a light microscope(ZEISS Axioplan 2) equipped with transmitted light micros-copy with a differential-interference contrast method and anAchroplan 40× objective. Cells incubated with DMSO and 12showed no fluorescence in comparison to the cells incubatedwith 13, which displayed intense fluorescent images (Figure 3).These results further confirm that 13 had improved the cellular

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uptake in comparison to 12, suggesting that fatty acylderivatives of FTC have better cellular uptake than FTC.

■ CONCLUSIONS

In summary, three fatty acyl conjugates of FTC weresynthesized as nucleoside prodrugs and were evaluated fortheir activities against various strains of HIV-1. The conjugationof FTC with selected long chain fatty acids improved the anti-HIV activity against both cell-free and cell-associated viruscompared to FTC alone and the corresponding physicalmixtures. 5′-O-Myristoyl FTC derivative 5 showed consistentlyhigh anti-HIV activity against cell-free (X4 and R5) strains andcell-associated virus, and was the most potent compoundamong previously studied similar fatty acyl esters of AZT, FLT,3TC, and d4T. Compound 5 was further tested against HIV-1clinical isolates, and antiviral activity was compared with that ofFTC. This compound exhibited significantly higher potencythan its parent nucleoside against wild-type and drug resistantclinical isolates. Cellular uptake studies confirmed that a fattyacyl ester analogue of FTC was accumulated intracellularly after1 h of incubation with CCRF-CEM cells and underwentintracellular hydrolysis, generating the parent nucleoside andfatty acid. All the fatty acyl ester conjugates of FTC showedhigher lipophilicity, as shown by calculated Lop P values. Thehigher lipophilicity and cellular uptake of fatty acyl esterconjugates of FTC would explain the higher anti-HIV activityversus FTC and the corresponding physical mixtures, and theirimproved resistance profile. 5′-O-Myristoyl FTC derivative 5was more potent and displayed a better resistance profile thanthe lead NRTI for therapy and prevention, tenofovir (data notshown). These FTC ester conjugates have the potential to bedeveloped as NRTI prodrugs, especially for topical anti-HIVmicrobicide applications.

■ ASSOCIATED CONTENT

*S Supporting InformationCharacterization of final compounds using 1H NMR and 13CNMR. This material is available free of charge via the Internetat http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*G.F.D.: Department of Obstetrics and Gynecology, EasternVirginia Medical School, 601 Colley Avenue, Norfolk, Virginia23507, USA; tel, +1-757-446-8908; fax, +1-757-446-9889; e-mail, DoncelGF@evms.edu. K.P.: 7 Greenhouse Road, Depart-ment of Biomedical and Pharmaceutical Sciences, College ofPharmacy, University of Rhode Island, Kingston, Rhode Island02881, USA; tel, +1-401-874-4471; fax, +1-401-874-5787; e-mail, kparang@uri.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

Support for this subproject (MSA-03-367) was provided byCONRAD, Eastern Virginia Medical School, under aCooperative Agreement (GPO-A-8-00-08-00005-00) with theUnited States Agency for International Development (USAID).The views expressed by the authors do not necessarily reflectthe views of USAID or CONRAD.

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