Emtricitabine Prodrugs with Improved Anti-HIV Activity and Cellular

Aug 23, 2012 - *G.F.D.: Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507, USA; tel...
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Emtricitabine Prodrugs with Improved Anti-HIV Activity and Cellular Uptake Hitesh 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) were synthesized and evaluated against HIV-1 cell-free and cellassociated virus and compared with the corresponding parent nucleoside and physical mixtures of FTC and fatty acids. Among all the compounds, the myristoylated conjugate of FTC (5, EC50 = 0.07−3.7 μM) displayed the highest potency. Compound 5 exhibited 10−24 and 3−13-times higher antiHIV activity than FTC alone (EC50 = 0.7−88.6 μM) and the corresponding 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 of incubation and underwent intracellular hydrolysis in CCRF-CEM cells. Alternative studies were conducted using the carboxyfluorescein conjugated with FTC though β-alanine (12) and 12-aminododecanoic acid (13). Acylation of FTC with a long-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) against multidrug resistant viruses B-NNRTI and B−K65R, indicating that FTC conjugation with myristic acid generates a more potent analogue with a better resistance profile than its parent compound. KEYWORDS: anti-HIV, (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine, cellular uptake, cytotoxicity, fatty acids

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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) and hepatitis B virus (HBV).1,2 Thus, FTC is clinically used as an anti-HIV agent in combination with other drugs in highly active antiretroviral therapy (HAART) and in treatment of HBV infection.1 FTC was developed as a 5-fluoro derivative of 3TC [lamivudine, (−)-2′,3′-dideoxy-3′-thiacytidine] and displayed 4−10 times more potency than 3TC against HIV-1.1−3 Due to structural similarities, FTC and 3TC share a common mechanism of action and drug resistance patterns.4 FTC is known to have a better therapeutic index than other similar NRTIs, such as 3TC, 3′-azido-2′,3′-dideoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).1,5−7 A single point mutation at residue methionine 184 to valine or isoleucine in HIV genome drastically reduces the activity of FTC against mutant virus (M184 V/I).8−10 Two major reasons for the drug resistance are cytidine deamination for both FTC and 3TC and the generation of steric hindrance by isoleucine substitution for methionine.8−10 Other mutant viral strains are also known to decrease the antiviral activity for 3TC and FTC such as mutation of methionine 552 to valine and isoleucine (M552 V/I) in HBV.1,11 Some studies suggest that FTC © 2012 American Chemical Society

generates a higher barrier to drug resistance and displays good synergism in combination with tenofovir than 3TC.12−14 Furthermore, nucleoside analogues often suffer from poor oral bioavailability because of their hydrophilic nature and limited cellular permeaibility.15 Several nucleoside prodrugs have been designed to enhance cellular uptake of these polar compounds.15 The polar nature of FTC (calculated Log P for FTC = −1.29) limits its efficient cellular uptake. We have previously reported the synthesis and evaluation of fatty acyl ester prodrugs of 3TC, AZT, d4T, and FLT.5,16−18 These studies showed improvement in the anti-HIV activities of nucleoside analogues after conjugation with myristic acid analogues.5,6,16−18 In addition, myristic acid analogues have moderate activity against N-myristoyltransferase (NMT), a crucial enzyme involved in the life cycle of HIV (e.g., capsid protein p17, Pr160gag‑pol, Pr55gag, p27nef).19,20 NMT catalyzes the myristoylation of viral proteins at N-terminal glycine and makes them more hydrophobic to improve their protein− Special Issue: Prodrug Design and Target Site Activation Received: Revised: Accepted: Published: 467

June 29, 2012 July 31, 2012 August 23, 2012 August 23, 2012 dx.doi.org/10.1021/mp300361a | Mol. Pharmaceutics 2013, 10, 467−476

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protein and protein−membrane interactions.20 Several myristic acid analogues inhibit NMT,21−23 thereby reducing replication of HIV-1. Our previous studies indicate that the conjugation of three fatty acids, myristic acid, 12-azidododecanoic acid, and 12-thioethyldodecanoic acid, with NRTIs generates more potent analogues.5,16−18 Herein, we discuss the synthesis and anti-HIV activity of fatty acyl esters of FTC as nucleoside prodrugs. The selection of the fatty acids, myristic acid, 12-azidododecanoic acid, and 12thioethyldodecanoic acid, was based on previous results about fatty acyl derivatives of other nucleosides.5,16−18 We hypothesized that the FTC conjugation with the myristic acid analogues would enhance the cellular uptake through improved lipophilicity. The fatty acyl esters are expected to get hydrolyzed intracellularly to produce two anti-HIV active agents, the nucleoside analogue and the fatty acid targeting RT and NMT enzymes, respectively. Higher uptake into HIV target cells and sustained intracellular release of two active agents would result in increased antiviral potency and higher barrier to development of drug resistance. These compounds were envisioned as improved prodrug nucleosides for microbicide development. Microbicides are topically applied agents designed to prevent or reduce transmission of sexually transmitted infections (STIs), in particular HIV/AIDS.24

according to the previously reported method.6 Compound 1 (250 mg, 0.45 mmol), the corresponding fatty acid (0.90 mmol), and HBTU (350 mg, 0.90 mmol) were dissolved in dry DMF (10 mL). DIPEA (2 mL, 15 mmol) was added to the reaction mixture, and stirring was continued overnight at room temperature. The reaction mixture was concentrated at reduced pressure, and the residue was purified by reversed phase HPLC using C18 column and water/acetonitrile as solvents as described above to afford 2−4. (−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradecanoyl)-5-fluoro-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%. 1 H NMR (400 MHz, CDCl3, δ ppm): 8.20−9.00 (br s, 1H, 4NH), 8.07 (d, J = 6.1 Hz, 1H, H-6), 7.22−7.33 (m, 5H, DMTr protons), 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, DMTrOCH3), 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 (br m, 14H, methylene protons). 13C NMR (CDCl3, 100 MHz, δ ppm): 173.12 (COO), 158.62 (C-4), 156.47 (C-2 CO), 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 (C1′), 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 (methylene carbons). 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%. 1 H NMR (400 MHz, CDCl3, δ ppm): 8.50−9.40 (br s, 1H, 4NH), 8.09 (d, J = 5.8 Hz, 1H, H-6), 7.23−7.34 (m, 5H, DMTr protons), 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, DMTrOCH3), 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 = 7.4 Hz, 2H, CH 2 CO), 1.52−1.72 (m, 4H, SCH 2 CH 2 , CH2CH2CO), 1.23−1.43 (br m, 17H, methylene protons). 13 C NMR (CDCl3, 100 MHz, δ ppm): 173.09 (COO), 158.63 (C-4), 156.23 (C2 CO), 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′-thiacytidine (5), (−)-5′-O-(12-Azidododecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (6), and (−)-5-Fluoro-5′-O-(12-thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacytidine (7). Acetic acid (80%, 10 mL) was added to compounds 2−4 (0.3 mmol). The reaction mixture was heated at 80 °C for 30 min. The reaction mixture was concentrated at reduced pressure, and the residue was purified by reversed phase HPLC using C18 column and water/acetonitrile as solvents as described above to yield 5−7.

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EXPERIMENTAL SECTION Materials and Methods. Emtricitabine (FTC) was purchased from Euro Asia Trans Continental (Bombay, India). 12-Bromododecanoic acid was purchased from Sigma Aldrich Chemical Co. 5(6)-Carboxyfluorescein (FAM) was purchased from Novabiochem. All the other reagents including solvents were purchased from Fisher Scientific. The final products were purified on a PhenomenexGemini 10 μm ODS reversed-phase column (2.1 × 25 cm) with a Hitachi HPLC system using a gradient system at a constant flow rate of 17 mL/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 a gradient system (water:acetonitrile) at constant flow rate of 1 mL/min with a UV detection at 265 nm (Table S2, Supporting Information). The chemical structures of final products were characterized by nuclear magnetic resonance spectrometry (1H NMR and 13 C NMR) determined on a Bruker NMR spectrometer (400 MHz) and confirmed by a high-resolution PE Biosystems Mariner API time-of-flight electrospray mass spectrometer. Chemical shifts are reported in parts per millions (ppm). For cellular uptake studies, cells were analyzed by flow cytometry (FACSCalibur: Becton Dickinson) using FITC channel and CellQuest software. Cell-viability studies were conducted using Cellometer Auto T.4 (Nexcelom Biosciences). The real time microscopy in live CCRF-CEM cell line with or without compounds was imaged using a ZEISS Axioplan 2 light microscope equipped with transmitted light microscopy with a differential-interference contrast method and an Achroplan 40× objective. Chemistry. (−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (2), (−)-5′-O-(12Azidododecanoyl)-N4-(4,4′-dimethoxytrityl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (3), and (−)-(4,4′-Dimethoxytrityl)-5′O-(12-thioethyldodecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (4). N4-DMTr protected FTC (1) was synthesized 468

dx.doi.org/10.1021/mp300361a | Mol. Pharmaceutics 2013, 10, 467−476

Molecular Pharmaceutics

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(−)-5′-O-(Tetradecanoyl)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine (5). Yield: 120 mg, 87%. 1H NMR (400 MHz, CDCl3, δ ppm): 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, H5″), 4.43 (dd, J = 12.6 and 2.2 Hz, 1H, H-5′), 3.56 (dd, J = 12.6 and 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 CO), 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 = 5.9 Hz, 1H, H-6), 6.25−6.29 (m, 1H, H-1′), 5.33−5.37 (m, 1H, H4′), 4.62 (dd, J = 12.6 and 4.0 Hz, 1H, H-5″), 4.43 (dd, J = 12.6 and 1.8 Hz, 1H, H-5′), 3.56 (dd, J = 12.6 and 5.2 Hz, 1H, H2″), 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 = 16.0 Hz, C-4), 151.89 (C-2 CO), 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′-dideoxy-3′-thiacytidine (7). Yield: 110 mg, 80%. 1H NMR (400 MHz, 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 and 4.1 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 and 2.1 Hz, 1H, H-2′), 2.49−2.58 (m, 4H, CH2SCH2), 2.30−2.45 (m, 2H, CH 2 COO), 1.53−1.69 (m, 4H, SCH 2 CH 2 , CH2CH2CO), 1.24−1.42 (br m, 17H, methylene protons). 13 C NMR (CDCl3, 100 MHz, δ ppm): 173.08 (COO), 156.00 (J = 17.2 Hz, C-4), 150.87 (C-2 CO), 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 (ESITOF) (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 (−)-5Fluoro-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dimethoxytrityl)-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 in a mixture of dry DMF (10 mL) and DIPEA (2 mL, 15 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated and dried under reduced pressure to afford crude 5′-O-Fmoc-amino acid derivatives 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)-N 4 (4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (9). HRMS (ESI-TOF) (m/z): C56H61FN4O8S, calcd, 968.4194; found, 991.4431 [M + Na]+. (−)-5-Fluoro-5′-O-(3-aminopropanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (10) and (−)-5-Fluoro5′-O-(12-aminododecanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′dideoxy-3′-thiacytidine (11). The crude products were dissolved in piperidine (20% in DMF, 10 mL), and the reaction mixture was stirred for 1 h at room temperature. The reaction solution was concentrated at reduced pressure. The residue was purified with reversed phase HPLC using C18 column and water/acetonitrile as solvents as described above to yield 10 and 11. (−)-5-Fluoro-5′-O-(3-aminopropanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (10). Overall yield: 200 mg, 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′-dimethoxytrityl)-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 and 12.2 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 and CH2CH2CO), 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 CO), 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). A mixture of 5(6)-carboxyfluorescein (430 mg, 1.15 mmol), the corresponding N4-DMTr-5′-O-aminoacyl derivative of FTC (10 or 11, 0.29 mmoL), and HBTU (440 mg, 1.15 mmol) was dissolved in a mixture of dry DMF (10 mL) and DIPEA (2 mL, 15 mmol) and stirred overnight at room temperature. The reaction mixture was concentrated and dried under vacuum. Acetic acid (80%, 10 mL) was added to the reaction mixture and was heated at 80 °C for 30 min. The final compounds (12 and 13) were purified with reversed phase HPLC using C18 column and using water/acetonitrile as solvents as described above. (−)-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 6 isomer), 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, FAMAr-H), 6.08−6.14 and 6.14−6.23 (two m, 1H, H-1′), 5.47 and 469

dx.doi.org/10.1021/mp300361a | Mol. Pharmaceutics 2013, 10, 467−476

Molecular Pharmaceutics

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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 and 12.6 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). 13C NMR (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 CO), 141.27, 137.72 (Ar-C-FAM), 137.08 (J = 238.5 Hz, C-5), 134.99, 131.01, 130.92, 129.33, 129.06, 128.72 (Ar-CFAM), 126.41 (J = 32.3 Hz, C-6), 121.50, 115.14, 112.07 and 112.04, (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, 0.17 H, FAM-Ar-H, 5 or 6 isomer) 8.49 (s, 0.63 H, FAM-Ar-H, 5 or 6 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 = 11.7 and 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 4.0 Hz, 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, 100 MHz, 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-2 CO), 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 (C1′), 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-1 and 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-free HIV-1 laboratory strains IIIB or BaL (Advanced Biotechnologies, Inc., Columbia, MD) in the absence or presence of each agent. After 2 h, cells were washed, cultured for an additional 46 h, and subsequently assayed for HIV-1 infection using the Galacto-Star β-Galactosidase Reporter Gene Assay System for Mammalian Cells (Applied Biosystems, Bedford, MA). Reductions in infection were calculated as a percentage relative to the level of infection in the absence of agents, and 50% inhibitory concentrations (EC50) were derived from regression analysis. Each compound concentration was tested in triplicate wells. Cell toxicity was evaluated using the same experimental design but without the addition of virus. The impact of compounds 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 [C subtype]) and drug resistant (clones = 4755-5, 71361-1, and A17) HIV-1 clinical isolates, phytohemagglutinin stimulated peripheral blood mononuclear cells (PBMCs) from at least two normal donors were pooled, diluted in fresh media, and plated in the interior wells of a 96 well round-bottom microplate. Each plate contained virus/cell control wells (cells + virus), experimental wells (drug + cells + virus), and compound control wells (drug + media, no cells, necessary for MTS monitoring of cytotoxicity). Test drug dilutions were prepared in microtiter tubes, and each concentration was placed in appropriate wells. Following addition of the drug dilutions to the PBMCs, a predetermined dilution of virus stock was then placed in each test well (final MOI ≅ 0.1). Since HIV-1 is not cytopathic to PBMCs, the same assay plate can be used for both antiviral efficacy and cytotoxicity measurements. Compounds were incubated with the virus and cells in a 96 well format for 6 h. 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 additional 150 μL of fresh medium was added to each well and further incubated for 6 days or until peak reverse transcriptase (RT) activity was detected. A microtiter plate-based RT reaction was utilized.28 Incorporated radioactivity (counts per minute, CPM) was quantified using standard liquid scintillation techniques. Compound IC 50 (50%, inhibition of virus replication) was calculated using statistical software and regression analysis. Cellular Uptake Studies. All of the stock solutions for compounds FAM, 5, 12, and 13 were prepared in DMSO. The human T lymphoblastoid cells CCRF-CEM (ATCC No. CCL119) were grown on 25 cm2 cell culture flasks with RMPI-1640 medium containing 10% fetal bovine serum. Upon reaching about 70% confluency, the cells were treated as described below and incubated for 1−24 h or longer at 37 °C. Cellular Uptake of 5 at Different Time Points. Cellular uptake and accumulation of compound 5 was evaluated in CCRF-CEM cells by analytical HPLC studies. CCRF-CEM cells were grown in 75 cm2 culture flasks with serum free RPMI medium to ∼70−80% confluency (∼1 × 106 cells/mL). The medium was replaced with RPMI medium containing compound 5 (50 μM). The cells were incubated at 37 °C for 1 h, 12 h, and 24 h. After incubation for the indicated time, the cells were collected by centrifugation. The medium was removed carefully by decantation, and the cell pellets were washed with ice-cold PBS to remove any medium. The cell pellets were thoroughly extracted with an equal volume of methanol, chloroform, and isopropanol mixture (100 μL, 4:3:1 v/v/v) and filtered through 0.2 μm filters. Compound 5 in cell lysates was detected by analytical HPLC analysis (20 μL injection) 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 for compound 5 was 18.2 min with this gradient system. The area under the curve for 5 after 1 h, 12 h, and 24 h was normalized per million cells, and the values (AUC/million cells) were compared. Cellular Uptake Study of Fluorescence-Labeled Nucleoside Analogues (12 and 13). All of the stock solutions for 470

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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 5 6 7 14 15 DMSO C-2i

FTC 5′-O-myristoyl FTC 5′-O-(12-azidododecanoyl) FTC 5′-O-(12-thioethyldodecanoyl) FTC myristic acid + FTC (50:50) 12-thioethyldodecanoic acid + FTC (50:50) dimethyl sulfoxide dextran sulfate

>200 >200 >200 93.6 >200 >200 >1000 >25

1.9 0.1 0.8 0.05 1.3 0.2 >1000 0.02

0.7 0.07 0.2 0.04 0.2 0.4 >1000 0.4

88.6 3.7 9.1 4.9 20 19.3 >1000 0.1

−1.29 5.96 5.17 5.05 NDh ND ND ND

a Data 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 partition coefficient 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)

in flow cytometry buffer, and the flow cytometry assays were performed 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 of cells to make the final concentration 10 μM. The cells were incubated for 1 h at 37 °C. Then the cells were treated with 0.25% trypsin/0.53 mM EDTA at room temperature for 5 min to remove any artificial uptake on the cell surface followed by washing with PBS (pH 7.4) three times. Washed cells were

compounds FAM, 12, and 13 were prepared in DMSO. The human T lymphoblastoid cells CCRF-CEM (ATCC No. CCL119) were grown on 25 cm2 cell culture flasks with RMPI-1640 medium containing 10% fetal bovine serum. When the cells reached about 70% confluency, FAM, 12, or 13 (1 mL, 20 μM) in RMPI-1640 medium was added to 1 mL of cells to make the final concentration 10 μM. The cells were incubated for 1 h at 37 °C. After 1 h of incubation, the cells were collected by centrifugation at 2000 rpm for 5 min. The cells were then washed with PBS (pH 7.4) three times. They were resuspended 471

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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)

IC90b (nM)

CC50c (nM)

TId

FTC

94US3393IN 98USMSC5016 A-17 MDR 71361-1 MDR 4755-5 MDR 94US3393IN 98USMSC5016 A-17 MDR 71361-1 MDR 4755-5 MDR

B C B-NNRTI B-K65R B-MDR B C B-NNRTI B-K65R B-MDR

12.1 2.8 35.1 16.0 5966.6 12144 6.6 10.9 15.7 16.1 >6561

>12144 >12144 >12144 >12144 >12144 >6561 >6561 >6561 >6561 >6561

>1004 >4337 346 >759 >2 >9373 >5965 4101 >4374 >3.8

5

a

IC50: 50% inhibitory concentration. bIC90: 90% inhibitory concentration. cTC50: 50% cytotoxic concentration. dTI: Therapeutic index. Multiple round 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 12 h, and (D) incubation for 24 h (x axis = minutes and y axis = mAU).

resuspended in flow cytometry buffer and the flow cytometry assays were performed as described below. Flow Cytometry. The cells were analyzed by flow cytometry (FACS Calibur: Becton Dickinson) using FITC channel and CellQuest software. The data presented are based on the mean fluorescence signal for 10000 cells collected. All the assays were done in triplicate. Real Time Fluorescence Microscopy in the Live CCRF-CEM Cell Line. The cellular uptake studies and intracellular localization of CCRE-CEM cells alone or incubated with 12 and 13 were imaged using a ZEISS Axioplan 2 light microscope equipped with transmitted light microscopy with a differentialinterference 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-1640 medium containing 10% fetal bovine serum. Upon reaching about 70% confluency, the cells were incubated with a solution of 10 μM of 12 and 13 for 1 h at 37 °C. They were then

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

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RESULTS AND DISCUSSION Chemistry. 5′-O-Fatty Acyl Ester Derivatives of FTC. 5′-OFatty acyl esters of FTC (5−7) were synthesized (Scheme 1)

Figure 2. Cellular uptake studies for 12 and 13 with DMSO and FAM as controls with and without treatment with trypsin. 472

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

corresponding N-Fmoc protected amino acid amides of FTC (8 and 9). The N-Fmoc groups were deprotected using crude reaction mixtures of 8 and 9 in the presence of piperidine to produce amino acid amides of FTC (10 and 11). Finally, FAM was attached to the free amino group in the presence of HBTU and DIPEA, followed by N4-DMTr deprotection in the presence 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 conjugate carboxyfluorescein through β-alanine (12) was used as a control FTC analogue whereas FTC attached to FAM through 12-aminododecanoyl (13) was used as an analogue of (−)-5fluoro-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 against cell-free, cell-associated, and multidrug resistant HIV-1. The antiviral activity of the fatty acyl ester derivatives of FTC is described in Table 1, and the results are compared with the parent nucleoside FTC alone and its physical mixtures with the corresponding fatty acids. The synthesized conjugates 5 and 6, and FTC, displayed no cytotoxicity at the highest tested concentrations (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

by the conjugation of fatty acids, myristic acid, 12azidododecanoic acid, and 12-thioethyldodecanoic acid with FTC. 5′-O-Substitution and selection of fatty acids were based on our recently published work on the conjugation of similar fatty acids with 3TC.16 5′-O-Fatty acyl derivatives of FTC (5−7) were synthesized from N4-DMTr protected FTC (1), which was synthesized according to the previously reported method.6 Compound 1 was reacted with the fatty acids (Scheme 1) in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) in dimethylformamide at room temperature, overnight, to produce (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 were purified by semipreparative HPLC. The purity of final products (>95%) was confirmed by analytical HPLC (Table S1, Supporting Information). The conjugation of the long chain fatty acids to FTC increased their partition coefficients (Log P) (Table 1), resulting in enhanced lipophilicity in comparison with the parent nucleoside analogue. 5(6)-Carboxyfluorescein Conjugates of FTC. 5(6)-Carboxyfluorescein (FAM) was conjugated with FTC using two different spacers, β-alanine and 12-aminododecanoic acid (Scheme 2). Initially, FTC-DMTr (1) was reacted with the corresponding N-Fmoc-amino acids in the presence of HBTU and DIPEA to produce DMTr-protected FTC conjugates of the 473

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to FTC alone. In order to better understand the cellular uptake profile of the FTC conjugates, the studies were conducted for 5 using human T lymphoblastoid cells (CCRF-CEM, ATCC No. CCL-119). Cells were grown to 70% confluency in the culture media 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 column chromatography and detection at 265 nm (see Materials and Methods 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 a significant decrease in the intracellular levels of 5. The results suggest that the conjugate was hydrolyzed intracellularly to FTC in the presence of intracellular esterases over the period of time. FTC and peaks of potentially phosphorylated products were observed at 1.7−2.9 min in the HPLC profile, suggesting intracellular hydrolysis. Conjugate 5 disappeared completely in the cellular extracts after 24 h. A strong peak was detected at 1.7−2.9 min, indicating the presence of metabolic products of 5. It is possible that FTC and its phosphorylated forms overlapped with cell extract peaks. These results suggest that conjugate 5 was able to cross the cell membrane and was hydrolyzed intracellularly as required for a prodrug to become active. However, overlapping peaks for FTC and the phosphorylated products with other intracellular cellular contents make this method inappropriate for comparative studies with the parent analogue. Thus, fluorescence-labeled fatty acyl analogues of FTC were synthesized for comparative studies. For a direct comparison between FTC and the FTC conjugates, cellular uptake studies were performed using FAM conjugates of FTC. FTC was attached to FAM through β-alanine (12) and 12-aminododecanoic acid (13) as the analogues of FTC and 6, respectively. CCRF-CEM cells were grown to 70% confluency in the culture media before they were incubated with the fluorescein-substituted conjugates, 12 and 13 (10 μM), for 1 h either in the presence or in the absence of trypsin (Figure 2). DMSO and FAM were used as controls for the study. Incubated cells were analyzed by flow cytometry (FACSCalibur: Becton Dickinson) using FITC channel and CellQuest software. The data are presented as the mean fluorescence signal for 10000 cells collected. The results clearly indicated that the presence of a long chain spacer between FAM and FTC improved the cellular uptake. Cellular uptake of 13 was 20-fold higher than that of 12. To confirm that the enhanced uptake of 13 was not just due to the outer cell membrane adherence, half of the cells from each batch were treated with trypsin for 5 min to wash the adsorbed molecules from the plasma cell membrane. The cellular uptake of 13 for trypsin treated cells was 8.5-fold higher than that of 12. Results suggest that a great proportion of cellular uptake of 13 was governed by improved lipophilicity and was not due to adsorption to the cell membrane. Real Time Fluorescence Microscopy in Live CCRF-CEM Cells. CCRF-CEM cells were incubated with DMSO, 12, and 13 (10 μM) for 1 h and imaged using a light microscope (ZEISS Axioplan 2) equipped with transmitted light microscopy with a differential-interference contrast method and an Achroplan 40× objective. Cells incubated with DMSO and 12 showed no fluorescence in comparison to the cells incubated with 13, which displayed intense fluorescent images (Figure 3). These results further confirm that 13 had improved the cellular

exhibited higher anti-HIV activity than FTC (Table 1). The FTC esters displayed the highest anti-HIV activity against cellfree virus (EC50 = 0.04−0.2 μM) among all the previously reported fatty acyl ester derivatives and their parent NRTIs.5,16−18 Myristic acid conjugate of FTC (5) showed high anti-HIV activity against cell-free virus (EC50 = 0.07−0.1 μM), which was ∼10−19 times better than that of FTC alone (EC50 = 0.7−1.9 μM). In addition, the anti-HIV activity against cell-associated virus for 5 (EC50 = 3.7 μM) was ∼24 times higher than that of FTC (EC50 = 88.6 μM). The 5′-O-12-thioethyldodecanoyl derivative of FTC (7) displayed slightly higher anti-HIV activity than 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 further compared with their corresponding physical mixtures. The equimolar (50:50) physical mixture of FTC with myristic acid (14) and 12-thioethyldodecanoic acid (15) showed significantly less potency (EC 50 = 0.2−20 μM) than the corresponding 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, approximately 3−13 times higher active than the corresponding physical mixture 14 (EC50 = 0.2−20 μM) against both cell-free and cell-associated virus (Table 1). These data suggest that the cellular uptake of the conjugates and intracellular hydrolysis to parent analogue and fatty acid contribute significantly to the higher anti-HIV profile of the ester conjugates versus the physical mixtures, and therefore, it is critically important to the improvement of viral inhibition. The anti-HIV activity of the physical mixtures was slightly higher than that of FTC. Comparative evaluation of FTC with the 14 and 15 indicated that the physical mixing produced a cooperative/additive antiHIV effect possibly through targeting two independent enzymes 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 of FTC (Table 2). The results indicated that myristoyl conjugation with FTC (5) improved the anti-HIV activity of FTC 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 5and 15-fold higher than that of FTC (IC90 = 32.4 and 161.9 nM). When tested against drug resistant viruses bearing mutations that confer resistance to nonnucleoside reverse transcriptase inhibitors (B-NNRTI) and the lead NRTI tenofovir (B-K65R), the IC90 values of 5 were 15.7 nM and 16.1 nM, respectively, being 6.6 and 35.2 times lower than those of FTC (IC90 = 103 and 567 nM). Analogue 5 was better than FTC even when compared to the multidrug resistant virus 4755-5, which confers resistance to numerous RTIs. The lower IC50 and IC90 values for 5 in comparison to FTC increased its therapeutic index. These results suggest that fatty acyl ester conjugate 5 has improved antiviral potency against wild-type and 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 chain fatty acids enhances the lipophilicity of the nucleoside derivatives. Previous studies on fatty acyl conjugates of d4T and 3TC indicated that fatty acyl conjugation resulted in improved lipophilicity and cellular uptake of nucleoside conjugates. Therefore, 5′-O-fatty acyl conjugates of FTC were also expected to have improved cellular uptake in comparison 474

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

CONCLUSIONS In summary, three fatty acyl conjugates of FTC were synthesized as nucleoside prodrugs and were evaluated for their activities against various strains of HIV-1. The conjugation of FTC with selected long chain fatty acids improved the antiHIV activity against both cell-free and cell-associated virus compared to FTC alone and the corresponding physical mixtures. 5′-O-Myristoyl FTC derivative 5 showed consistently high anti-HIV activity against cell-free (X4 and R5) strains and cell-associated virus, and was the most potent compound among previously studied similar fatty acyl esters of AZT, FLT, 3TC, and d4T. Compound 5 was further tested against HIV-1 clinical isolates, and antiviral activity was compared with that of FTC. This compound exhibited significantly higher potency than its parent nucleoside against wild-type and drug resistant clinical isolates. Cellular uptake studies confirmed that a fatty acyl ester analogue of FTC was accumulated intracellularly after 1 h of incubation with CCRF-CEM cells and underwent intracellular hydrolysis, generating the parent nucleoside and fatty acid. All the fatty acyl ester conjugates of FTC showed higher lipophilicity, as shown by calculated Lop P values. The higher lipophilicity and cellular uptake of fatty acyl ester conjugates of FTC would explain the higher anti-HIV activity versus FTC and the corresponding physical mixtures, and their improved resistance profile. 5′-O-Myristoyl FTC derivative 5 was more potent and displayed a better resistance profile than the lead NRTI for therapy and prevention, tenofovir (data not shown). These FTC ester conjugates have the potential to be developed as NRTI prodrugs, especially for topical anti-HIV microbicide applications. ASSOCIATED CONTENT

S Supporting Information *

Characterization of final compounds using 1H NMR and 13C NMR. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*G.F.D.: Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia 23507, USA; tel, +1-757-446-8908; fax, +1-757-446-9889; email, [email protected]. K.P.: 7 Greenhouse Road, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, USA; tel, +1-401-874-4471; fax, +1-401-874-5787; email, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this subproject (MSA-03-367) was provided by CONRAD, Eastern Virginia Medical School, under a Cooperative Agreement (GPO-A-8-00-08-00005-00) with the United States Agency for International Development (USAID). The views expressed by the authors do not necessarily reflect the views of USAID or CONRAD. 475

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