Synthesis and Antiviral Evaluation of 3′Branched Nucleoside Analogs

Arch. Pharm. Chem. Life Sci. 2008, 341, 9 –19 N. R. El-Brollosy et al. 9

Full Paper

Synthesis and Antiviral Evaluation of 6-(Trifluoromethylbenzyl)and 6-(Fluorobenzyl) Analogues of HIV Drugs Emivirine andGCA-186

Nasser R. El-Brollosy1, 2, Esben R. Sørensen1, Erik B. Pedersen1, Giuseppina Sanna3,Paolo La Colla3, and Roberta Loddo3

1 Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense M,Denmark

2 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, SaudiArabia

3 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale eBiotecnologie Microbiche, Universita di Cagliari, Monserrato, Italy

The present study describes the synthesis and antiviral evaluation of a series of novel 6-(3-tri-fluoromethylbenzyl) and 6-(fluorobenzyl) analogues of the HIV drugs emivirine and GCA-186.The objective was to investigate whether the fluoro or trifluoromethyl substituents could leadto an improved antiviral activity against HIV-1 wild type and mutants resistant to non-nucleo-side RT inhibitors. The biological test results showed that the most of theses compounds showedgood activity against wild type HIV-1. Among them, compound 1-(ethoxymethyl)-6-(3-fluoroben-zyl)-5-isopropyluracil (9i) showed the largest inhibitory potency (EC50 = 0.02 lM), resultingequally potent than emivirine against wild type HIV-1. Furthermore, compound 9i showed mar-ginal better activity against resistant mutants than emivirine. The key steps in the synthesis ofthe target compounds were either reaction of an appropriate b-keto ester with thiourea or across-coupling reaction of 6-chloro-2,4-dimethoxypyrimidines with benzylic Grignard reagents.

Keywords: Alkenyloxymethyluracils / Grignard reaction / HIV-1 / Non-nucleoside reverse transcriptase inhibitors /

Received: May 30, 2007; accepted: August 24, 2007

DOI 10.1002/ardp.200700113

Introduction

The reverse transcriptase (RT) enzyme of the humanimmunodeficiency virus type 1 (HIV-1) is an importanttarget for development of anti-AIDS drugs. It is responsi-ble for the conversion of a single-stranded RNA viralgenome into a double-stranded DNA chain that subse-

quently is incorporated into the DNA of the infected hostcell [1, 2]. In recent years, much effort has been put intothe design and synthesis of HIV-1 non-nucleoside RTinhibitors (NNRTIs) [3]. The NNRTIs are highly specific astheir binding site is a hydrophobic pocket locatedapproximately 10 � from the polymerease active site [4]forcing the RT subunit into an inactive conformation [5].One of the first NNRTIs was 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) [6, 7]. Although HEPT didnot show very high activity against HIV-1, it was consid-ered an interesting lead compound for the synthesis ofnew analogues. Among them 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (Emivirine, formerly MKC-442) [8], 6-(3,5-dimethylbenzyl)-5-isopropyluracil (GCA-186) [9], andthe corresponding N-1 allyloxymethyl analogues [10](BED-60 and AMB-A10) showed high potent activityagainst HIV-1 (Fig. 1).

Correspondence: Erik B. Pedersen, Nucleic Acid Center, Department ofPhysics and Chemistry, University of Southern Denmark, Campusvej 55,DK-5230 Odense M, Denmark.E-mail: [email protected]: +45 6615 8780

Abbreviations: reverse transcriptase (RT); human immunodeficiency vi-rus type 1 (HIV-1); non-nucleoside RT inhibitors (NNRTIs); 1-[(2-hydrox-yethoxy)methyl]-6-(phenylthio)thymine (HEPT); N,O-bis(trimethylsilyl)a-cetamide (BSA); trimethylsilyl trifluoro-methanesulfonate (TMS triflate); 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT)

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10 N. R. El-Brollosy et al. Arch. Pharm. Chem. Life Sci. 2008, 341, 9 –19

During NNRTI mono therapy for HIV-1 infectedpatients [11] and in-vitro culture [12], rapid emergence ofhighly drug-resistant viruses is often observed. The pri-mary cause of drug resistance is the substitution of tyro-sine by cysteine at position 181 in the HIV-1 RT (Y181C).Furthermore, the RT mutation of the lysine at position103 to asparagine (K103N) is frequently observed on treat-ment with retroviral regimens containing many NNRTIs[13] including the FDA-(U.S. Food and Drug Administra-tion) approved drugs (nevirapine [14], delavirdine [15],and efavirenz [16]). Therefore, it is of importance to findnovel NNRTIs that may be able to overcome such resist-ance issue.

GCA-186 (Fig. 1) differs structurally from emivirineonly in the introduction of two methyl substituents in 3-and 5-position at the C-6 benzyl group. It performed bet-ter in tolerating the presence of the Y181C and K103Nmutations than did emivirine itself. The increase ofpotency of GCA-186 over emivirine against the wildtypeHIV and mutants has been correlated with an overalltighter and/or more complete fit in the hydrophobicpocket due to the presence of 3,5-dimethyl groups [9]. Onthe other hand, the two methyl substituents on GCA-186were thought to undergo metabolism, e.g. by the cyto-chrome P 450 family in the liver.

Recently, we synthesized a series of 6-(3,5-dichloroben-zyl) derivatives as isosteric analogues of GCA-186 [17] on atrial to avoid rapid metabolism in the liver. In an effort toimprove the activity against HIV-1 wild type and resistantmutants, and as a part of our continuing interest in thechemistry of NNRTIs [10, 17–21], the present studydescribes the synthesis and antiviral evaluation of 6-(tri-fluoromethylbenzyl) and 6-(fluorobenzyl) analogues ofemivirine and GCA-186. The objective was to investigatewhether the fluoro- or trifluoromethyl substituentscould lead to an improved antiviral activity against HIV-1

wildtype and mutants resistant to non-nucleoside RTinhibitors (NNRTIs).

Results and discussion

ChemistryThe required key intermediate – the 5,6-disubstituteduracil derivatives 4a–i were synthesized via two inde-pendent routes based on the commercial availability ofthe starting materials (Scheme 1).

With the nitriles commercially available for com-pound 1a, b, we choose the b-keto ester strategy, devel-oped by one of us [22], for the synthesis of the correspond-ing uracil derivatives 4a–d (Scheme 2). However, withthe fluoro-substituted benzylic bromide commerciallyavailable for compounds 7a–d, we choose another strat-egy based on a Grignard reaction followed by a cross-cou-pling reaction for the synthesis of the uracil derivatives4e–i (Scheme 3). This method was chosen despite the factthat it is known that benzylic-type Grignard reagents canbe difficult to prepare via the classical method with thetreatment of magnesium powder.

(Trifluoromethylphenyl)acetonitriles 1a, b werereacted with the zinc organometallic reagent from ethyl2-bromobutyrate or methyl 2-bromoisovalerate in anhy-drous THF [22] to give the corresponding b-keto esters 2a-d which were condensed with thiourea in the presence ofsodium ethoxide to afford the 2-thiouracils 3a–d. TheNMR spectra of the crude compounds 2a, c showed animpurity identified as another b-keto ester resultingfrom self-condensation of ethyl 2-bromobutyrate. Onreaction with thiourea, the b-keto ester impurity alsoformed a pyrimidine compound as an impurity in theraw materials of 3a, c which were easily purified accord-

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Figure 1. Structures of the lead molecules HEPT, MKC-442,GCA-186, BED-60, and AMB-A10.

Scheme 1. Synthetic route for uracils 4a– i.

Arch. Pharm. Chem. Life Sci. 2008, 341, 9 –19 New Emivirine and GCA-186 Analogues 11

ing to usual work-up procedures [23]. De-sulfurization ofcompounds 3a–d with boiling aqueous chloroacetic acidgave the corresponding uracils 4a–d (Scheme 2).

The fluoro-substituted uracil analogues 4e–i were syn-thesized in analogy to literature procedures [24, 25]. Theappropriate Grignard reagents synthesized in situ in astandard way from the corresponding bromide asdescribed by Wakefield [24] for benzyl bromide, wereadded to the methoxy-protected uracil derivatives 6a [26]or 6b [25] in the presence of an appropriate catalyst. A sys-tematic investigation (including use of different cata-lysts, solvents, reaction time, and temperature) showedthat the best results were obtained when Fe(acac)3 wasused as a catalyst in the cross-coupling reaction and thesubsequent hydrolysis for the compounds with the fol-lowing aromatic substitution pattern: 3-F (8a/4e, 61%),2,5-F (8b/4f, 24%), and 2,3,5,6-F (8d/4h, 18%). The signifi-cant drop in yields, corresponding to the number andpositions, was occupied by the fluoro group on the aro-mat with the highest yield for the mono-substituted aro-mat. Surprisingly, it was not possible to synthesize com-pound 8c using Fe(acac)3 as a catalyst. However, we suc-ceeded to synthesize compound 8c in 10% yield usingcommercially available catalyst NiCl2(dppe). It is note-worthy that the catalyst used by Adriane et al. [25] –NiCl2(tpp) [27] was not successful in our hands.

It was not possible to isolate the pure compounds 8a–edue to a complex mixture. Nevertheless, compounds 4e–i were easily obtained as solid compounds after hydroly-sis of the corresponding methoxy-protected compounds8a–e in boiling aqueous HCl following evaporation andadition of water with overall yields of 8–61% (Scheme 3).The low yield for the synthesis of 4i (8%) compared to thecorresponding 5-ethyl derivative 4e obtained in 61% yieldis presumably due to steric hindrance of the larger iso-propyl at the 5-position of the uracil ring. However, the

cross-coupling reaction was responsible for the overalllow yield as the Grignard reagents were obtained inquantitative yield according to TLC analysis showing dis-appearance of the benzyl bromide.


(a) R9CH(Br) CO2R2, Zn, THF; (b) K2CO3; (c) HCl, 90 – 94% (one-pot synthesis);(d) (H2N)2CS, NaOEt, 19 – 60%; (e) aq ClCH2CO2H 61 – 72%.

Scheme 2. Synthetic route of compounds 1–4.

(a) Ref. [25, 26]; (b) 4 equiv. Mg powder, dry Et2O; (c) 6a or 6b, cat., dry Et2O; (d) aq.HCl (4M).


(a) BSA, H3CCH2OCH2Cl, CsI, CHCl3, 19 – 95%; (b) BSA, TMS-triflate, R2CH2O-CH2OCH2R2, H3CCN, 38 – 89%.



The 1-ethoxymethyluracils 9a–i were prepared from4a–i via silylation in situ with N,O-bis(trimethylsilyl)ace-tamide (BSA) in anhydrous chloroform followed by treat-ment with chloromethyl ethyl ether in the presence ofcesium iodide to give the MKC-442 analogues 9a-i in 19–95% yields (Scheme 4). For the synthesis of compounds10–12, the method was modified. The appropriate ura-cils 4a–f or 4h were silylated in situ with BSA in anhy-drous acetonitrile and alkylated at the N-1 position withbis(allyloxy)methane [10, 28] (for compounds 4a–f, and4h) or bis(propargyloxy)methane [10] (for compounds4a–c, 4e, f, and 4h) or bis(2-methylallyoxy)methane [10](for compounds 4e, f and 4h) in the presence of trimethyl-silyl trifluoro-methanesulfonate (TMS triflate) [29] as a

Lewis acid catalyst to give the target molecules; 1-(ally-oxymethyl)uracils 10a–f, and 10h, 1-(propargyloxyme-thyl)uracils 11a–c, 11e, f, and 11h and 1-(2-methylally-oxymethyl)uracils 12e, f, and 12h, respectively(Scheme 4). The yield for the alkylation reactions variedin a range from 38 to 89%. Especially, the propynyloxy-methyl alkylation proceeded badly, even though the reac-tion time was extended to three days. However, we suc-ceeded to increase the yield up to 81% by using fourequivalents of TMS triflate, and the reaction was finishedafter about 4 h.

The compounds were identified by comparison of simi-lar NMR data [10, 18–23]. MS and elemental analysis arein full agreement with the proposed structures.


Table 1. Antiviral activity and cytotoxicity of compounds 9–12 against HIV-1 in MT-4 cells.a)

Compound Wild type N119 (Y181C) A17 (K103N +Y181C)

EFVR (K103R +V179D + P225H)

EC50b) (lM) CC50

c) (lM) SId) EC50b) (lM) EC50

b) (lM) EC50b) (lM)

9a9b9c9d9e9f9g9h9i10a10b10c10d10e10f10h11a11b11c11e11f11h12e12f12hMKC-442BED-60AMB-A10GCA-186Efavirenz

0.4 l 0.10.1 l 0.081.9 l 0.70.9 l 0.50.05 l 0.010.05 l 0.0010.08 l 0.020.09 l 0.0050.02 l 0.0050.2 l 0.10.05 l 0.031.7 l 0.71.3 l 0.10.03 l 0.0050.04 l 0.020.07 l 0.010.2 l 0.10.05 l 0.033 l 10.05 l 0.010.05 l 0.0050.09 l 0.0050.1 l 0.010.2 l 0.050.4 l 0.050.03 l 0.0050.004 l 0.0050.009 l 0.0010.0006 [8]0.002 l 0.0005

A10054 l 3A100A100A100A100A100A100A10077 l 539 l 0.5A100A100A100A100A10068 l 546 l 4A100A100A1005 l 1A10056 l 238 l 1A100855543 [8]30 l 1

A250240A52A111A2000A2000A1250A1111A5000385780A59A77A3333A2500A142834092033A2000A200056A100028095A33332125061117200015000

4.5 l 0.31.5 l 0.58017 l 318 l 211 l 0.5352314 l 33.2 l 0.51.3 l 0.2A10013 l 110 l 114 l 6405.5 l 0.52.6 l 0.8100 l 3A2017 l 3A52425 l 5A38A200.3 l 0.010.4 l 0.06

0.008 l 0.0009

45 l 1512 l 2A100A100685087544219 l 912 l 4A100A1004214 l 644A6810 l 0.5A1005653A5A100A56A38562.0 l 0.36.3 l 1.4

0.3 l 0.05

A100A54A100A100A100A100A100A10056A77A39A100A1006070A100A68A46A100A100A100A5A100A56A38A1006.2 l 0.36.1 l 0.7

3.0 l 1

a) Data represent mean values of at least two separate experiments.b) Compound dose required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the

MTT method.c) Compound dose required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.d) Selectivity index: ratio CC50/ED50. The symbol (>) indicates that CC50 was not reached at the highest concentration tested. For

description of assay see Experimental, section 4.


Biological screeningCompounds 9a–i and 10–12 were tested for biologicalactivity against wildtype HIV-1 strain IIIB in MT-4 cells.Results were compared with the antiviral activity of emi-virine, a well examined HEPT analogues and efavirenz,the most active anti-HIV drug used in therapy today.Compounds were also tested against NNRTI-resistantmutants [Y181C, K103N + Y181C, and triple mutant(K103R + V179D + P225H), highly resistant to efavirenz].Results are listed in Table 1.

As seen from the results listed in Tabel 1, most of thetested compounds showed good activity against wildtypeHIV-1 with a wide range of EC50 values from 3–0.02 lM.Generally, the majority of the novel compounds were notcytotoxic for MT-4 cells at the maximum concentrationtested (100 lM). The only exception is compound 11h,which was surprisingly cytotoxic (5 lM). In general, the3-fluoro-substituted uracil series (9e, 10e, and 11e) were10-times more potent than the corresponding CF3-series(9a, 10a, and 11a). Compound 9i, the most active com-pound tested against HIV-1, substituted at R by a fluorineatom was in our study marginally more potent thanMKC-442 (20 nM versus 30 nM). Increasing the electrone-gativity by the replacements of hydrogen atoms with upto four fluorine atoms on the benzyl ring, is well toler-ated (9f–h, 10h, and 11f, h). However, replacement of the3,5-dimethyl groups in BED-60, with the strong with-drawing group CF3 (9c, 10c, and 11c) resulted in the leastactive among the synthesized compounds. In fact, theactivity was about 100-times lower when compared withthe corresponding 3,5-dichloro derivatives which havepreviously been synthesized in our laboratory as isostericanalogues of GCA-186 and BED-60. Furthermore, theintroduction of different unsaturated N-1 substituentshad no effect on the anti-HIV-1 activity, except, whenincreasing the bulkiness around the double bond of 9ewith methyl moieties (12e–h), leads to a significant dropin activity compared to 9e (2-, 4-, and 8-fold, respectively)which is in in accordance with our previous observations[10]. It is well known that introduction of an isopropylgroup instead of an ethyl group at C-5 leads to a rise inbiologic activity. Surprisingly, only the emivirine ana-logues having an allyloxymethyl or a propynyloxymethylat the N-1 position, and substitution with one CF3 groupon the aromatic ring leads to an up to 4-fold increase inactivity (11a vs. 11b and 10a vs. 10b).

Unfortunately, only a marginally improvement inactivity compared to the clinically important mutants(N119, A17 or EFVR) was observed for compounds 9a, b,10a, b and 11a, b when compared with MKC-442.

Conclusion

In summary, we have described the synthesis and anti-HIV activity of novel 6-(3-trifluoromethylbenzyl) and 6-(fluoromethylbenzyl) analogues of emivirine, GCA-186,and BED-60. The most active compounds 9i and 10eshowed activity against wildtype HIV-1 in the nanomolarrange with a selective index of greater than 3333.

This work received funding from the European Community'sSixth Framework Programme under contract number LSHP-CT-2004-503162 (Selection and development of microbicides formucosal use to prevent sexual HIV transmission/acquisition)[30].

The authors have declared no conflict of interest.

Experimental

ChemistryNMR spectra were recorded on a Varian Gemini 2000 NMR spec-trometer (Varian Inc., Palo Alto, CA, USA ) at 300 MHz for 1H and75 MHz for 13C with TMS as internal standard. Due to complexityof 13C-NMR spectra for polyfluorinated benzylic groups, onlystrong peaks are reported for the benzylic group. MALDI spectrawere recorded on a Fourier Transform (FT) Ion Cyclotron Reso-nance Mass Spectrometer (IonSpec Corporation, Lake Forest, CA,USA). Melting points were determined on a B�chi melting pointapparatus (B�chi Labortechnik, Flawil, Switzerland). Elementaryanalyses were performed at H.C. Ørsted Institute, University ofCopenhagen. Silica gel (0.040-0.063 mm) used for column chro-matography and analytical silica gel TLC plates 60 F254 were pur-chased from Merck (Darmstadt, Germany). Solvents used for col-umn chromatography were distilled prior to use. The fluoro-ben-zyl bromides were purchased from Fluorochem,8 Old Glossop,Derbyshire, UK) Mg powder was purchased from Riedel-De Ha�n(Seelze, Germany). Reagents were used as purchased.

General procedure for synthesis of 2-alkyl-4-aryl-3-oxobutyrates 2a–dZn dust 25 –30 g was activated by stirring with 4 M HCl (100 mL)for 5 min. The zinc dust was filtered and washed sequentiallywith water, ethyl alcohol, and ether. Dried by evaporation underreduced pressure at 808C for five hours, and kept in vacuo over-night. The activated Zn was suspended in anhydrous THF(35 mL) and heated to reflux. A few drops of ethyl 2-bromobuty-rate or methyl 2-bromoisovalerate were added and the mixturewas refluxed for 10 min. The appropriate nitrile; (3-trifluorome-thylphenyl)acetonitrile 1a or [3,5-bis(trifluoromethyl)phenyl]a-cetonitrile 1b (0.015 mol) was added in one portion and ethyl 2-bromobutyrate or methyl 2-bromoisovalerate (0.03 mol) wasadded dropwise. After the addition was completed, the mixturewas refluxed for 30 min. The reaction mixture was diluted withTHF (100 mL) and saturated aqueous K2CO3 (30 mL) was added.The mixture was stirred for 1 h and the THF layer was decanted



off. The residue was washed with THF (3620 mL) and the com-bined THF fractions were stirred with 10% aqueous HCl (20 mL)for 30 min. The solution was concentrated under reduced pres-sure and diluted with CH2Cl2 (75 mL). The organic phase waswashed with saturated aqueous NaHCO3 (2650 mL), dried(Na2SO4), and evaporated under reduced pressure to give 2a –das pale yellow oils.

Ethyl-2-ethyl-3-oxo-4-(3-trifluoromethylphenyl)butyrate2aYield 4.1 g (91%). 1H-NMR (CDCl3): d (ppm) = 0.80 (t, 3H, J = 7.2 Hz,CH3), 1.16 (t, 3H, J = 7.1 Hz, CH3), 1.78 (m, 2H, CH2), 3.37 (t, 1H, J =7.3 Hz, CH), 3.81 (s, 2H, CH2), 4.07 (q, 2H, J = 7.2 Hz, CH2), 7.30 –7.47 (m, 4H, Harom). 13C-NMR (CDCl3): d (ppm) = 11.77 (CH3), 14.01(CH3), 21.51 (CH2), 48.21 (CH2), 60.06 (CH), 61.45 (CH2), 123.98,124.03, 126.32, 126.37, 128.99, 133.07, 134.25 (CF3, Carom), 169.43(CO), 201.80 (CO). HRMS – MALDI: m/z = 325.1022 [M+Na+](C15H17F3O3Na+); requires 325.1030.

Methyl-2-isopropyl-3-oxo-4-(3-trifluoromethyl-phenyl)butyrate 2bYield 4.3 g (94%). 1H-NMR (CDCl3): d (ppm) = 0.89, 0.96 (26d, 6H, J= 6.9 Hz, 26CH3), 2.43–2.49 (m, 1H, CH), 3.31 (d, 1H, J = 7.3 Hz,CH), 3.69 (s, 3H, OCH3), 3.87 (s, 2H, CH2), 7.36–7.54 (m, 4H, Harom).13C-NMR (CDCl3): d (ppm) = 20.35 (CH3), 20.53 (CH3), 28.70 (CH),48.61 (CH2), 52.24 (OCH3), 66.15 (CH), 124.02, 124.07, 126.37,126.42, 128.99, 133.13, 134.09 (CF3, Carom), 169.27 (CO), 201.44(CO). HRMS – MALDI: m/z = 325.1022 [M+Na+] (C15H17F3O3Na+);requires 325.1025.

Ethyl-4-[3,5-bis(trifluoromethyl)phenyl]-2-ethyl-3-oxobutyrate 2cYield 5.1 g (92%). 1H-NMR (CDCl3): d (ppm) = 0.92 (t, 3H, J = 7.4 Hz,CH3), 1.25 (t, 3H, J = 7.1 Hz, CH3), 1.90 (m, 2H, CH2), 3.46 (t, 1H, J =7.4 Hz, CH), 3.96 (s, 2H, CH2), 4.18 (q, 2H, J = 7.1 Hz, CH2), 7.64 (s,2H, Harom), 7.79 (s, 1H, Harom). 13C-NMR (CDCl3): d (ppm) = 11.82(CH3), 14.03 (CH3), 21.55 (CH2), 47.54 (CH2), 60.45 (CH), 61.65(CH2), 121.20, 129.95, 131.52, 131.97, 135.79 (CF3, Carom), 169.35(CO), 201.01 (CO).

Methyl-4-[3,5-bis(trifluoromethyl)phenyl]-2-isopropyl-3-oxobutyrate 2dYield 5.0 g (90%). 1H-NMR (CDCl3): d (ppm) = 0.94, 0.99 (26d, 6H, J= 6.9 Hz, 26CH3), 1.97 (m, 1H, CH), 3.33 (d, 1H, J = 7.3 Hz, CH),3.73 (s, 3H, OCH3), 3.97 (s, 2H, CH2), 7.63 (s, 2H, Harom), 7.79 (s, 1H,Harom). 13C-NMR (CDCl3): d (ppm) = 20.38 (CH3), 20.56 (CH3), 28.87(CH), 47.79 (CH2), 52.37 (OCH3), 66.52 (CH), 121.22, 129.97,131.52, 131.96, 135.65 (CF3, Harom), 169.24 (CO), 200.73 (CO).

General procedure for synthesis of 5-alkyl-6-(trifluoromethyl)benzyl-2-thiouracils 3a–dNa (4.92 g, 0.217 mol) was dissolved in absolute ethanol(100 mL). Thiourea (11.42 g, 0.15 mol) was added and the reac-tion mixture was heated to reflux. The b-keto ester 2a–d(0.01 mol) was added dropwise and the mixture was refluxed fortwo hours. The mixture was evaporated to dryness underreduced pressure and the residue was redissolved in H2O(100 mL). The solution was acidified with HCl to pH 4. The pre-

cipitate thus formed was filtered off, washed with H2O, thenwith Et2O, and dried to give 3a –d as white solids.

5-Ethyl-6-(3-trifluoromethylbenzyl)-2-thiouracil 3aYield 1.88 g (60%); mp. 182 –1848C. 1H-NMR (DMSO-d6): d (ppm) =0.80 (t, 3H, J = 7.2 Hz, CH3), 2.28 (q, 2H, J = 7.2 Hz, CH2), 3.97 (s, 2H,CH2), 7.53 –7.67 (m, 4H, Harom), 12.29, 12.41 (26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 12.77 (CH3), 17.69 (CH2), 34.13 (CH2),117.41 (C-5), 123.47, 123.52, 124.95, 125.00, 129.71, 132.09,138.21 (CF3, Carom), 148.27 (C-6), 161.35 (C-4), 174.24 (C-2). MS-EI:m/z (%) = 314 [M+] (100).

5-Isopropyl-6-(3-trifluoromethylbenzyl)-2-thiouracil 3bYield 1.9 g (58%); mp. 189-1918C. 1H-NMR (DMSO-d6): d (ppm) =0.85 (d, 6H, J = 6.6 Hz, 26CH3), 2.85 (hept. 1H, J = 6.6 Hz, CH), 4.01(s, 2H, CH2), 7.50 –7.64 (m, 4H, Harom), 12.23, 12.36 (26s, 2H,26NH). 13C-NMR (DMSO-d6): d (ppm) = 19.45 (26CH3), 26.57 (CH),34.25 (CH2), 119.85 (C-5), 123.34, 123.48, 124.68, 124.73, 129.73,132.09, 138.31 (CF3, Carom), 148.02 (C-6), 160.61 (C-4), 174.20 (C-2).MS-EI: m/z (%) = 328 [M+] (100).

6-[3,5-Bis(trifluoromethyl)benzyl]-5-ethyl-2-thiouracil 3cYield 1.75 g (46%); mp. 223 –2258C. 1H-NMR (DMSO-d6): d (ppm) =0.80 (t, 3H, J = 7.4 Hz, CH3), 2.14 (q, 2H, J = 7.4 Hz, CH2), 4.08 (s, 2H,CH2), 8.01 (s, 2H, Harom), 8.03 (s, 1H, Harom), 12.28, 12.47 (26s, 2H,26NH). 13C-NMR (DMSO-d6): d (ppm) = 12.76 (CH3), 17.68 (CH2),33.90 (CH2), 117.83 (C-5), 120.68, 124.99, 129.20, 130.12, 140.21(CF3, Carom), 147.23 (C-6), 161.33 (C-4), 174.33 (C-2). MS-EI: m/z (%) =382 [M+] (29).

6-[3,5-Bis(trifluoromethyl)benzyl]-5-isopropyl-2-thiouracil3dYield 0.75 g (19%); mp. 210 –2128C. 1H-NMR (DMSO-d6): d (ppm) =1.09 (d, 6H, J = 6.6 Hz, CH3), 2.94 (hept. 1H, J = 6.6 Hz, CH), 4.10 (s,2H, CH2), 7.99 (s, 2H, Harom), 8.02 (s, 1H, Harom), 12.22, 12.37 (26s,2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 19.43 (CH3), 26.56 (CH),34.01 (CH2), 120.57 (C-5), 120.66, 125.03, 129.09, 130.61, 140.32(CF3, Carom), 147.05 (C-6), 160.58 (C-4), 174.29 (C-2). MS-EI: m/z (%) =396 [M+] (58).

General procedure for synthesis of 5-alkyl-6-(trifluoromethylbenzyl)uracils 4a–dA suspension of 2-thiouracils (3a–d, 0.01 mol) in 10% aqueousClCH2CO2H (200 mL) was refluxed overnight. After cooling, theprecipitate was filtered off, washed with cold EtOH, then Et2O,and dried to give 4a –d as white solids.

5-Ethyl-6-(3-trifluoromethylbenzyl)uracil 4aYield 2.1 g (70%); mp. 218-2198C. 1H-NMR (DMSO-d6): d (ppm) =0.91 (t, 3H, J = 6.5 Hz, CH3), 2.46 (q, 2H, J = 6.5 Hz, CH2), 3.87 (s, 2H,CH2), 7.36 –7.54 (m, 4H, Harom), 10.79, 11.12 (26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 13.39 (CH3), 17.55 (CH2), 34.54 (CH2),111.63 (C-5), 123.41, 123.46, 124.89, 124.94, 129.64, 132.14,138.35 (CF3, Carom), 147.73 (C-6), 150.86 (C-2), 164.38 (C-4). MS-EI:m/z (%) = 298 [M+] (66). Anal. Calcd. for C14H13F3N2O260.25 H2O: C,55.54; H, 4.49; N, 9.25. Found: C, 55.76; H, 4.24; N, 9.24.



5-Isopropyl-6-(3-trifluoromethylbenzyl)uracil 4bYield 2.27 g (73%); mp. 206 –2088C. 1H-NMR (DMSO-d6): d (ppm) =1.07 (d, 6H, J = 6.7 Hz, 26CH3), 2.83 (hept, 1H, J = 6.7 Hz, CH),3.89 (s, 2H, CH2), 7.58–7.64 (m, 4H, Harom), 10.85 (br s, 2H, 26NH).13C-NMR (DMSO-d6): d (ppm) = 19.99 (CH3), 26.36 (CH), 34.78 (CH2),114.20 (C-5), 123.37, 123.43, 124.62, 124.68, 129.69, 132.11,138.49 (CF3, Carom), 147.47 (C-6), 150.87 (C-2), 163.74 (C-4). MS-EI:m/z (%) = 312 [M+] (47). Anal. calcd for C15H15F3N2O260.25 H2O: C,56.87; H, 4.93; N, 8.84. Found: C, 56.94; H, 4.67; N, 8.82.

6-[3,5-Bis(trifluoromethyl)benzyl]-5-ethyluracil 4cYield 2.3 g (63%), mp. 250 –2528C. 1H-NMR (DMSO-d6): d (ppm) =0.81 (t, 3H, J = 7.2 Hz, CH3), 2.29 (q, 2H, J = 7.2 Hz, CH2), 3.99 (s, 2H,CH2), 8.02 –8.03 (m, 3H, Harom), 10.79, 11.08 (26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 13.39 (CH3), 17.52 (CH2), 34.28 (CH2),112.01 (C-5), 120.62, 125.01, 129.19, 130.54, 140.37 (CF3, Carom),146.78 (C-6), 150.84 (C-2), 164.32 (C-4). MS- EI: m/z (%) = 366 [M+](49). Anal. calcd for C15H12F6N2O2: C, 49.19; H, 3.30; N, 7.65.Found: C, 49.23; H, 3.16; N, 7.55.

6-[3,5-Bis(trifluoromethyl)benzyl]-5-isopropyluracil 4dYield 2.3 g (61%); mp. 231 –2338C. 1H-NMR (DMSO-d6): d (ppm) =1.10 (d, 6H, J = 6.8 Hz, 26CH3), 2.91 (hept, 1H, J = 6.8 Hz, CH),4.01 (s, 2H, CH2), 8.01 (s, 2H, Harom), 8.04 (s, 1H, Harom), 10.77, 10.98(26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 19.92 (CH3),26.33 (CH), 34.47 (CH2), 114.49 (C-5), 120.55, 125.03, 129.01,130.59, 140.51 (CF3, Carom), 146.55 (C-6), 150.84 (C-2), 163.67 (C-4).MS-EI: m/z (%) = 380 [M+] (58). Anal. calcd for C16H14F6N2O2: C,50.53; H, 3.71; N, 7.37. Found: C, 50.92; H, 3.59; N, 7.35.

General procedure for synthesis of 5-alkyl-6-(fluoromethylbenzyl)uracils 4e-iIn-vacuo dried magnesium powder (4 equiv.) and anhydrous Et2Owas stirred under a dry nitrogen atmosphere. A crystal of I2 wasadded. Immediately after addition of the first few drops of theappropriate fluorobenzyl bromide (1 equiv in Et2O), the colorturned from red/brown to white/green and the rest of the bro-mide was added dropwise during 10 min. The reaction mixturewas stirred for one hour at room temperature and used in thenext step without further purification.

A mixture of the methoxy protected uracil chloride 6a or 6b (1equiv.), Fe(acac)3 (0.1 equiv.) or NiCl2(dppe) (30 mol% based on 6b)and Et2O was stirred under a dry nitrogen atmosphere. A solu-tion of the appropriate fluorobenzylmagnesium bromide (1.33equiv.) in anhydrous Et2O was added via a syringe. During theaddition, the reaction mixture started to reflux and a color shiftfrom red to brown was observed. The stirring was continuedovernight at room temperature. The reaction mixture wasquenched with saturated NH4Cl, and extracted with H2O. Theaqueous layer was extracted with Et2O, and the combined etherphases were dried (MgSO4) and evaporated in vacuo to give a yel-low/brown oil. The crude products were deprotected in refluxed4 M HCl overnight. Upon cooling to room temperature, whitecrystals precipitated. These were filtered off, washed with Et2O,and dried in vacuo to give 4e – i.

5-Ethyl-6-(3-fluorobenzyl)uracil 4eYield 798 mg (61%); mp. 224 –2268C. 1H-NMR (DMSO-d6): d (ppm)= 0.84 (t, 3H, J = 7.3 Hz, CH3), 2.25 (q, 2H, J = 7.3 Hz, CH2), 3.79 (s,2H, CH2), 7.07 –7.43 (m, 4H, Harom), 10.73, 11.04 (26s, 2H,

26NH). 13C-NMR (DMSO-d6): d (ppm) = 13.43 (CH3), 17.53 (CH2),34.55 (CH2), 111,49 (C-5), 113.37 (d, J = 20.3 Hz, Carom), 115.00 (d, J= 21.8 Hz, Carom), 124.14 (d, J = 3.0 Hz, Carom), 130.46 (d, J = 9.0 Hz,Carom), 139.67 (d, J = 10.0 Hz, Carom), 147,91 (C-6), 150,85 (C-2),162.13 (d, J = 242.20 Hz, Carom), 164,40 (C-4). HRMS – MALDI: m/z =249.1034 [M + H+] (C13H13FN2O2

+); requires 249.1037.

5-Ethyl-6-(2,5-difluorobenzyl)uracil 4fYield 308 mg (24%); mp. 223 –2258C. 1H-NMR (DMSO-d6): d (ppm)= 0.80 (t, 3H, J = 7.2 Hz, CH3), 2.19 (q, 2H, J = 7.3 Hz, CH2), 3.79 (s,2H, CH2), 7.09 –7.31 (m, 3H, Harom), 10.71, 11.06 (26s, 2H,26NH). 13C-NMR (DMSO-d6): d (ppm) = 13.29 (CH3), 17.44 (CH2),28.25 (CH2), 112.17 (C-5), 114.91 –116.91, 125.54 –125.65 (Carom),146.42 (C-6), 150.82 (C-2), 156.34 (d, J = 239.3 Hz, Carom), 158.11 (d,J = 241.5 Hz, Carom), 164.31 (C-4). HRMS – MALDI: m/z = 267.0940[M+H+] (C13H12F2N2O2

+); requires 267.0940.

5-Ethyl-6-(2,3,6-trifluorobenzyl)uracil 4gYield 333 mg (10%); mp 215 –2208C. 1H-NMR (DMSO-d6): d (ppm) =0.68 (t, 3H, J = 7.2 Hz, CH3), 2.17 (q, 2H, J = 7.3 Hz, CH2), 3.88 (s, 2H,CH2), 7.12 –7.52 (m, 2H, Harom), 10.75, 11.05 (26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 12.66 (CH3), 17.33 (CH2), 23.95 (CH2),111.48 (C-5), 111.16 –116.47 (Carom), 146.68 (C-6), 150.69 (C-2),164.18 (C-4). HRMS – MALDI: m/z = 285.0845 [M+H+](C13H11F3N2O2

+); requires 285.0848.

5-Ethyl-6-(2,3,5,6-tetrafluorobenzyl)uracil 4hYield 690 mg (18%); mp. 205 –2108C. 1H-NMR (DMSO-d6): d (ppm)= 0.76 (t, 3H, J = 7.3 Hz, CH3), 2.19 (q, 2H, J = 7.3 Hz, CH2), 3.95 (s,2H, CH2), 7.86 (m, 1H, Harom), 10.73, 11.09 (26s, 2H, 26NH). 13C-NMR (DMSO-d6): d (ppm) = 12.79 (CH3), 17.36 (CH2), 23.98 (CH2),102.96 –106.00 (Carom), 111.75 (C-5), 145.89 (C-6), 150.68 (C-2),164.10 (C-4). HRMS – MALDI: m/z = 303.0751 [M+H+](C13H10F4N2O2

+); requires 303.0758.

6-(3-Fluorobenzyl)-5-isopropyluracil 4iYield 198 mg (8%). 1H-NMR (DMSO-d6): d (ppm) = 1.06, 1.08 (d, 6H,J = 6 Hz, 26CH3), 3.05 (sep, 1H, CH), 3.80 (s, 2H, CH2), 7.08–7.39(m, 4H, Harom), 10.71, 10.90 (26s, 2H, 26NH). 13C-NMR (DMSO-d6):d (ppm) = 19.32 (26CH3), 20.02 (CH), 31.49 (CH2), 113.41 (d, J =20.2 Hz, Carom), 114.86 (d, J = 21.0 Hz, Carom), 124.00 (d, J = 3.0 Hz,Carom), 130.47 (d, J = 8.2 Hz, Carom), 139.84 (d, J = 7.5 Hz, Carom),162.13 (d, J = 204.9 Hz, Carom), 114.10 (C-5), 147.64 (C-6), 150.84 (C-2), 162.19 (C-4).

General procedure for N-1 alkylation of 4a–iMethod A: To a suspension of compound 4a–i (1 equiv.), in anhy-drous CHCl3 (20 mL), was added N,O-bis(trimethylsilyl)acetamide(BSA) (3.5 equiv.) and the mixture was stirred under a nitrogenatmosphere at room temperature. After a clear solution wasobtained (10 min), chloromethyl ethyl ether (1.5 equiv.) and CsI(1 equiv.) were added. The reaction mixture was stirred at roomtemperature under a nitrogen atmosphere for three hours (forthe synthesis of compounds 9a–d) or overnight (for the synthe-sis of compounds 9e– i). The reaction mixture was quenchedwith saturated aqueous NaHCO3 (20 mL). The mixture wasextracted with CH2Cl2 (3650 mL). The organic phases were col-lected, dried (MgSO4), and evaporated under reduced pressure.The products were purified by silica gel column chromatogra-



phy using 0-1% MeOH/CHCl3 (9a–d) or 42% EtOH/petroleumether (9e – i) as eluents.

1-(Ethoxymethyl)-5-ethyl-6-(3-trifluoromethylbenzyl)uracil 9aYield 256 mg (72%) as white solid; mp. 131 –1328C. 1H-NMR(CDCl3): d (ppm) = 1.04 (t, 3H, J = 7.4 Hz, CH3), 1.13 (t, 3H, J =7.0 Hz, CH3), 2.42 (q, 2H, J = 7.4 Hz, CH2), 3.57 (q, 2H, J = 7.0 Hz,CH3), 4.22 (s, 2H, CH2), 5.11 (s, 2H, CH2), 7.23–7.56 (m, 4H, Harom).13C-NMR (CDCl3): d (ppm) = 13.67 (CH3), 14.94 (CH3), 19.21 (CH2),33.23 (CH2), 65.08 (CH2), 72.82 (CH2), 117.36 (C-5), 124.10, 124.18,124.23, 124.29, 129.73, 130.44, 136.47 (CF3, Carom), 147.93 (C-6),151.89 (C-2), 163.25 (C-4). MS-EI: m/z (%) = 356 [M+] (25). Anal. calcdfor C17H19F3N2O3: C, 57.30; H, 5.37; N, 7.86. Found: C, 57.16; H,5.32; H, 7.76.

1-(Ethoxymethyl)-5-isopropyl-6-(3-trifluoromethylbenzyl)uracil 9bYield 288 mg (78%) as white solid; mp. 138 –1398C. 1H-NMR(CDCl3): d (ppm) = 1.14 (t, 3H, J = 7.1 Hz, CH3), 1.28 (d, 6H, J =6.9 Hz, 26CH3), 2.75 (hept, 1H, J = 6.9 Hz, CH), 3.59 (q, 2H, J =7.1 Hz, CH2), 4.25 (s, 2H, CH2), 5.14 (s, 2H, CH2), 7.31 –7.57 (m, 4H,Harom), 9.41 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 14.93 (CH3),20.27 (CH3), 28.47 (CH), 33.31 (CH2), 65.09 (CH2), 72.98 (CH2),120.14 (C-5), 124.06, 124.11, 124.14, 124.19, 129.67, 130.54,136.61 (CF3, Carom), 147.36 (C-6), 151.84 (C-2), 162.23 (C-4). HRMS –MALDI: m/z = 393.1396 [M+Na+] (C18H21F3N2O3Na+); requires393.1404. Anal. calcd for C18H21F3N2O3: C, 58.37; H, 5.72; N, 7.56.Found: C, 58.33; H, 5.66; N, 7.53.

6-[3,5-Bis(trifluoromethyl)benzyl]-1-(ethoxymethyl)-5-ethyluracil 9cYield 300 mg (71%) as white solid; mp. 139 –1408C. 1H-NMR(CDCl3): d (ppm) = 1.04 (t, 3H, J = 7.4 Hz, CH3), 1.10 (t, 3H, J =7.1 Hz, CH3), 2.41 (q, 2H, J = 7.4 Hz, CH2), 3.56 (q, 2H, J = 7.1 Hz,CH2), 4.29 (s, 2H, CH2), 5.14 (s, 2H, CH2), 7.58 (s, 2H, Harom), 7.82 (s,1H, Harom), 9.53 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 13.57(CH3), 14.82 (CH3), 19.27 (CH2), 33.24 (CH2), 65.19 (CH2), 72.96(CH2), 117.81 (C-5), 121.15, 127.42, 127.46, 132.82, 138.31 (CF3,Carom), 146.75 (C-6), 151.67 (C-2), 162.92 (C-4). HRMS – MALDI: m/z= 447.1114 [M+Na+] (C18H18F6N2O3Na+); requires 447.1124. Anal.calcd for C18H18F6N2O3: C, 50.95; H, 4.28; N, 6.60. Found: C, 51.25;H, 4.28; N, 6.54.

6-[3,5-Bis(trifluoromethyl)benzyl]-1-(ethoxymethyl)-5-isopropyluracil 9dYield 306 mg (70%) as white solid; mp. 160 –1618C. 1H-NMR(CDCl3): d (ppm) = 1.09 (t, 3H, J = 7.1 Hz, CH3), 1.28 (d, 6H, J =6.8 Hz, 26CH3), 2.67 (hept, 1H, J = 6.8 Hz, CH), 3.58 (q, 2H, J =7.1 Hz, CH2), 4.31 (s, 2H, CH2), 5.18 (s, 2H, CH2), 7.60 (s, 2H, Harom),7.82 (s, 1H, Harom), 9.40 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) =14.81 (CH3), 20.16 (CH3), 28.66 (CH), 33.22 (CH2), 65.18 (CH2),73.12 (CH2), 120.56 (C-5), 121.33, 124.81, 127.45, 132.75, 138.39(CF3, Carom), 146.17 (C-6), 151.70 (C-2), 161.99 (C-4). HRMS –MALDI: m/z = 461.1270 [M+Na+] (C19H20F6N2O3Na+); requires461.1280.

1-(Ethoxymethyl)-5-ethyl-6-(3-fluorobenzyl)uracil 9eYield 117 mg (95%) as white solid; mp. 114 –1168C. 1H-NMR(CDCl3): d (ppm) = 1.05 (t, 3H, J = 7.5 Hz, CH3), 1.16 (t, 3H, J =

7.2 Hz, CH3), 2.44 (q, 2H, J = 7.5 Hz, CH2), 3.59 (q, 2H, J = 7.2 Hz,CH2), 4.14 (s, 2H, CH2), 5.10 (s, 2H, CH2), 6.81 –7.33 (m, 4H, Harom),9.59 (s, H, NH). 13C-NMR (CDCl3): d (ppm) = 13.71 (CH3), 14.98 (CH3),19.14 (CH2), 33.11 (CH2), 65.04 (CH2), 72.73 (CH2), 114.33 (d, J =21.0 Hz, Carom), 114.41 (d, J = 22.5 Hz, Carom), 122.93 (d, J = 3.0 Hz,Carom), 130.74 (d, J = 8.3 Hz, Carom), 137.80 (d, J = 7.5 Hz, Carom),163.24 (d, J = 246.0 Hz, Carom), 117.16 (C-5), 148.32 (C-6), 151.86(C-2), 164.24 (C-4). HRMS – MALDI: m/z = 329.1272 [M+Na+](C16H19FN2O3Na+); requires 329,1273. Anal. calcd for C16H19FN2O3:C, 62,73; H, 6,25; N, 9,14. Found: C, 62,85; H, 6,31; N, 9,12.

6-(2,5-Difluorobenzyl)-1-(ethoxymethyl)-5-ethyluracil 9fYield 206 mg (85%) as white solid; mp. 117–1208C. 1H-NMR(CDCl3): d (ppm) = 1.05 (t, 3H, J = 7.2 Hz, CH3), 1.15 (t, 3H, J =7.2 Hz, CH3), 2.44 (q, 2H, J = 7.2 Hz, CH2), 3.58 (q, 2H, J = 7.2 Hz,CH2), 4.12 (s, 2H, CH2), 5.14 (s, 2H, CH2), 6.62–7.12 (m, 3H, Harom),9.54 (s, H, NH). 13C-NMR (CDCl3): d (ppm) = 13.57 (CH3), 14.87(CH3), 19.12 (CH2), 29.71 (d, J = 4.5 Hz, CH2), 64.96 (CH2), 72.87(CH2), 114.45 –116.94, 124.35 –124,69, 156.36 (d, J = 220.8 Hz,Carom), 158.95 (d, J = 242.3 Hz, Carom), 117.63 (C-5), 147.31 (C-6),151.71 (C-2), 162.99 (C-4). HRMS – MALDI: m/z = 347.1178 [M+Na+](C16H18F2N2O3Na+); requires 347.1171. Anal. calcd forC16H18F2N2O3: C, 59.25; H, 5.59; N, 8.64. Found: C, 59.25; H, 5.50;N, 8.60.

1-(Ethoxymethyl)-5-ethyl-6-(2,3,6-trifluorobenzyl)uracil9gYield 5 mg (11%) as white solid. 1H-NMR (CDCl3): d (ppm) = 0.89 (t,3H, J = 7.4 Hz, CH3), 1.17 (t, 3H, J = 7.1 Hz, CH3), 2.37 (q, 2H, J =7.5 Hz, CH2), 3.60 (q, 2H, J = 7.1 Hz, CH2), 4.15 (s, 2H, CH2), 5.35 (s,2H, CH2), 6.82–7.15 (m, 2H, Harom), 8.53 (s, H, NH). 13C-NMR(CDCl3): d (ppm) = 12.78 (CH3), 14.94 (CH3), 19.21 (CH2), 23.27(CH2), 64.88 (CH2), 73.20 (CH2), 110.87–111.34, 115.79–117.10(Carom), 117.05 (C-5), 147.54 (C-6), 151.54 (C-2), 162.86 (C-4). HRMS– MALDI: m/z = 365.1084 [M+Na+] (C16H17F3N2O3Na+); requires365.1078.

1-(Ethoxymethyl)-5-ethyl-6-(2,3,5,6-tetrafluorobenzyl)uracil 9hYield 84 mg (36%). 1H-NMR (CDCl3): d (ppm) = 0.92 (t, 3H, J =7.5 Hz, CH3), 1.15 (t, 3H, J = 7.2 Hz, CH3), 2.38 (q, 2H, J = 7.5 Hz,CH2), 3.60 (q, 2H, J = 7.2 Hz, CH2), 4.20 (s, 2H, CH2), 5.35 (s, 2H,CH2), 6.82 –7.15 (m, 1H, Harom), 9.40 (s, H, NH). 13C-NMR (CDCl3): d

(ppm) = 12.81 (CH3), 14.89 (CH3), 19.19 (CH2), 23.47 (CH2), 64.95(CH2), 73.19 (CH2), 104.96–106.01 (Carom), 117.37 (C-5), 146.68 (C-6), 151.59 (C-2), 162.89 (C-4). HRMS – MALDI: m/z = 383.0989[M+Na+] (C16H16F4N2O3Na+); requires 383.0989.

1-(Ethoxymethyl)-6-(3-fluorobenzyl)-5-isopropyluracil 9iYield 23 mg (19%). 1H-NMR (CDCl3): d (ppm) = 1.18 (t, 3H, J =7.2 Hz, CH3), 1.28 (d, 6H, J = 6.9 Hz, 26CH3), 2.83 (sep, 1H, CH),3.61 (q, 2H, J = 7.2 Hz, CH2), 4.18 (s, 2H, CH2), 5.13 (s, 2H, CH2),6.82 –7.35 (m, 4H, Harom), 9.45 (s, H, NH). 13C-NMR (CDCl3): d (ppm)= 14.97 (CH3), 19.53 (CH), 20.33 (26CH3), 33.19 (CH2), 65.02 (CH2),72.88 (CH2), 114.33 (d, J = 21.0 Hz, Carom), 114.24 (d, J = 21.0 Hz,Carom), 114.37 (d, J = 21.8 Hz, Carom), 122.86 (d, J = 3.0 Hz, Carom),130.69 (d, J = 8.3 Hz, Carom), 137.99 (d, J = 7.5 Hz, Carom), 163.22 (d, J= 246.0 Hz, Carom), 119.90 (C-5), 147.77 (C-6), 151.81 (C-2), 162.36(C-4). HRMS – MALDI: m/z = 343.1428 [M+Na+] (C17H21FN2O3Na+);



requires 343.1427. Anal. calcd for C17H21FN2O360.5 H2O: C,61.99; H, 6.73; N, 8.59. Found: C, 61.58; H, 6.97; N, 8.86.

General procedure for N-1 alkylation of 4a-f, hMethod B: The pyrimidine 4a–f (1 equiv.) was stirred in anhy-drous CH3CN (15 mL) under a nitrogen atmosphere and BSA (3.5equiv.) was added. After a clear solution was obtained (10 min),the mixture was cooled to -508C, and TMS triflate (one equiv. forpreparation of 10a –f, 10h, and 12e, f, h; four equiv. for prepara-tion of 11a –c, 11e, f, and 11h) was added followed by dropwiseaddition of the appropriate acetal (2 equiv.). The reaction mix-ture was stirred at room temperature for 3–4 hours (for the syn-thesis of compounds 10a –d and 11a –c) or overnight (for the syn-thesis of compounds 10e, f, g, 11e, f, h, and 12e, f, h). The reac-tion was quenched with cold, satutated, aqueous NaHCO3 solu-tion (5 mL), and the solvent was evaporated under reduced pres-sure at room temperature. The residue was extracted withCH2Cl2 (3650 mL). The organic phases were collected, dried(MgSO4), and evaporated under reduced pressure. The productswere purified by silica gel column chromatography using 0–1%MeOH/CHCl3 (10a –d and 11a–c) or 42% EtOH/petroleum ether(10e, f, g, 11e, f, h, and 12e, f, h) as eluents.

1-(Allyloxymethyl)-5-ethyl-6-(3-trifluoromethylbenzyl)uracil 10aYield 253 mg (69%) as white foams. 1H-NMR (CDCl3): d (ppm) =1.04 (t, 3H, J = 7.5 Hz, CH3), 2.43 (q, 2H, J = 7.5 Hz, CH2), 4.11 (dd,2H, J = 1.3, 5.7 Hz, CH2), 4.23 (s, 2H, CH2), 5.13 (s, 2H, CH2), 5.17 –5.31 (m, 2H, CH2=), 5.77 –5.90 (m, 1H, =CH), 7.26–7.57 (m, 4H,Harom), 9.47 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 13.68 (CH3),19.21 (CH2), 33.28 (CH2), 70.59 (CH2), 72.57 (CH2), 117.45 (C-5),117.99 (CH2=), 124.20, 124.25, 124.31, 124.36, 129.79, 130.45,136.33 (CF3, Carom), 133.35 (=CH), 147.84 (C-6), 151.76 (C-2), 163.08(C-4). HRMS – MALDI: m/z = 391.1240 [M+Na+] (C18H19F3N2O3Na+);requires 391.1235.

1-(Allyloxymethyl)-5-isopropyl-6-(3-trifluoromethylbenzyl)uracil 10bYield 290 mg (76%) as white solid; mp. 112 –1138C. 1H-NMR(CDCl3): d (ppm) = 1.28 (d, 6H, J = 6.9 Hz, CH3), 2.77 (hept, 1H, J =6.9 Hz, CH), 4.11 (dd, 2H, J = 1.2, 4.4 Hz, CH2), 4.25 (s, 2H, CH2),5.16 (s, 2H, CH2), 5.17 –5.31 (m, 2H, CH2=), 5.79–5.86 (m, 1H,=CH), 7.30 –7.57 (m, 4H, Harom), 9.41 (s, 1H, NH). 13C-NMR (CDCl3):d (ppm) = 20.29 (CH3), 28.49 (CH), 33.36 (CH2), 70.57 (CH2), 72.74(CH2), 117.96 (C-5), 120.26 (CH2=), 124.08, 124.13, 124.21, 124.26,129.73, 130.53, 136.48 (CF3, Carom), 133.36 (=CH), 147.25 (C-6),151.83 (C-2), 162.20 (C-4). MS-EI: m/z (%) = 382 [M+] (5). Anal. calcdfor C19H21F3N2O3: C, 59.68; H, 5.54; N, 7.33. Found: C, 59.68; H,5.48; N, 7.34.

1-(Allyloxymethyl)-6-[3,5-bis(trifluoromethyl)benzyl]-5-ethyluracil 10cYield 296 mg (68%) as white solid; mp. 131 –1328C. 1H-NMR(CDCl3): d (ppm) = 1.05 (t, 3H, J = 7.4 Hz, CH3), 2.46 (q, 2H, J =7.4 Hz, CH2), 4.08 (d, 2H, J = 5.9 Hz, CH2), 4.30 (s, 2H, CH2), 5.16 (s,2H, CH2), 5.20–5.28 (m, 2H, CH2=), 5.74–5.87 (m, 1H, =CH), 7.58(s, 2H, Harom), 7.82 (s, 1H, Harom), 9.66 (s, 1H, NH). 13C-NMR (CDCl3):d (ppm) = 13.59 (CH3), 19.28 (CH2), 33.27 (CH2), 70.59 (CH2), 72.66(CH2), 117.93 (C-5), 118.17 (CH2=), 121.56, 124.74, 127.41, 132.86,138.15 (CF3, Carom), 133.06 (=CH), 146.63 (C-6), 151.68 (C-2), 162.94

(C-4). HRMS – MALDI: m/z = 459.1114 [M+Na+] (C19H18F6N2O3Na+);requires 459.1129. Anal. calcd for C19H18F6N2O3: C, 52.30; H, 4.16;N, 6.42. Found: C, 52.53; H, 4.13; N, 6.33.

1-(Allyloxymethyl)-6-[3,5-bis(trifluoromethyl)benzyl]-5-isopropyluracil 10dYield 298 mg (66%) as white solid; mp. 166–1678C. 1H-NMR(CDCl3): d (ppm) = 1.28 (d, 6H, J = 6.9 Hz, 26CH3), 2.70 (hept, 1H, J= 6.9 Hz, CH), 4.11 (d, 2H, J = 5.6 Hz, CH2), 4.31 (s, 2H, CH2), 5.16 –5.26 (m, 2H, CH2=), 5.18 (s, 2H, CH2), 5.74 –5.84 (m, 1H, =CH), 7.59(s, 2H, Harom), 7.82 (s, 1H, Harom), 9.29 (s, 1H, NH). 13C-NMR (CDCl3):d (ppm) = 20.18 (CH3), 28.68 (CH), 33.35 (CH2), 70.56 (CH2), 72.82(CH2), 118.16 (C-5), 120.66 (CH2=), 121.41, 124.79, 127.43, 132.81,138.21 (CF3, Carom), 133.06 (=CH), 146.06 (C-6), 151.61 (C-2), 161.91(C-4). HRMS – MALDI: m/z = 473.1270 [M+Na+] (C20H20F6N2O3Na+);requires 473.1283.

1-(Allyloxymethyl)-5-ethyl-6-(3-fluorobenzyl)uracil 10eYield 227 mg (89%) as white solid; mp. 60 –628C. 1H-NMR (CDCl3):d (ppm) = 1.07 (t, 3H, J = 7.4 Hz, CH3), 2.45 (q, 2H, J = 7.2 Hz, CH2),4.10 (dt, 2H, J = 5.7, 1.5 Hz, CH2), 4.16 (s, 2H, CH2), 5.13 (s, 2H,CH2), 5.17–5.32 (m, 2H, =CH2), 5.79 –5.90 (m, 1H, CH=), 6.82 –7.35 (m, 4H, Harom), 9.42 (s, H, NH). 13C-NMR (CDCl3): d (ppm) =13.72 (CH3), 19.16 (CH2), 33.16 (CH2), 70.56 (CH2), 72.54 (CH2),117.87 (=CH2), 133.45 (CH=), 114.40 (d, J = 21.0 Hz, Carom), 122.94(d, J = 3.0 Hz, Carom), 130.75 (d, J = 8.3 Hz, Carom), 137.64 (d, J =6.8 Hz, Carom), 163.26 (d, J = 246.0 Hz, Carom), 117.24 (C-5), 148.23(C-6), 151.77 (C-2), 163.12 (C-4). HRMS – MALDI: m/z = 341,1277[M+Na+] (C17H19FN2O3Na+); requires 341,1310. Anal. calcd forC17H19FN2O3: C, 64.14; H, 6.02; N, 8.80. Found: C, 64.22; H, 6.05; N,8.75.

1-(Allyloxymethyl)-6-(2,5-difluorobenzyl)-5-ethyluracil 10fYield 165 mg (84%) as white solid; mp. 93–968C. 1H-NMR (CDCl3):d (ppm) = 1.05 (t, 3H, J = 7.5 Hz, CH3), 2.41 (q, 2H, J = 7.5 Hz, CH2),4.09 (dt, 2H, J = 5.4, 1.2 Hz, CH2), 4.13 (s, 2H, CH2), 5.15 (s, 2H,CH2), 5.17–5.30 (m, 2H, =CH2), 5.78 –5.90 (m, 1H, CH=), 6.62 –7.12 (m, 3H, Harom), 9.42 (s, H, NH). 13C-NMR (CDCl3): d (ppm) =13.59 (CH3), 19.15 (CH2), 26.77 (d, J = 3.8 Hz, CH2), 70.48 (CH2),72.66 (CH2), 117.89 (=CH2), 133.29 (CH=), 114.52–117.00,124.31 –124.55, 156.37 (d, J = 243.8 Hz, Carom), 159.00 (d, J =243.0 Hz, Carom), 117.71 (C-5), 147.23 (C-6), 151.61 (C-2), 162.85 (C-4). HRMS – MALDI: m/z = 359,1178 [M+Na+] (C17H18F2N2O3Na+);requires 359,1180. Anal. calcd for C17H18F2N2O3: C, 60.71; H, 5.39;N, 8.33. Found: C, 60.73; H, 5.27; N, 8.25.

1-(Allyloxymethyl)-5-ethyl-6-(2,3,5,6-tetrafluorobenzyl)uracil 10hYield 115 mg (48%). 1H-NMR (CDCl3): d (ppm) = 1.04 (t, 3H, J =7.5 Hz, CH3), 2.49 (q, 2H, J = 7.5 Hz, CH2), 4.15 (dt, 2H, J = 5.4,1.2 Hz, CH2), 4.20 (s, 2H, CH2), 5.41 (s, 2H, CH2), 5.14 –5.34 (m, 2H,=CH2), 5.76 –5.95 (m, 1H, CH=), 7.00 –7.15 (m, 1H, Harom), 9.00 (s,H, NH). 13C-NMR (CDCl3): d (ppm) = 13.22 (CH3), 18.77 (CH2), 24.03(CH2), 69.92 (CH2), 71.31 (CH2), 117.85 (=CH2), 133.25 (CH=),105.02-106.09, 117.21–117.45 (Carom), 114.14 (C-5), 146.56 (C-6),151.45 (C-2), 162.75 (C-4). HRMS – MALDI: m/z = 395,0989 [M+Na+](C17H16F4N2O3Na+); requires 395,0971.

5-Ethyl-1-(prop-2-ynyloxymethyl)-6-(3-trifluoromethylbenzyl)uracil 11aYield 297 mg (81%) as white solid; mp. 129 –1308C. 1H-NMR(CDCl3): d (ppm) = 1.05 (t, 3H, J = 7.5 Hz, CH3), 2.44 (t, 1H, J =



2.4 Hz, CH:), 2.45 (q, 2H, J = 7.5 Hz, CH2), 4.22 (s, 2H, CH2), 4.28(d, 2H, J = 2.4 Hz, CH2), 5.18 (s, 2H, CH2), 7.27 –7.58 (m, 4H, Harom),9.64 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 13.72 (CH3), 19.19(CH2), 33.21 (CH2), 57.16 (CH2), 72.51(CH:), 74.92 (CH2), 78.85(:C), 117.59 (C-5), 124.25, 124.29, 124.35, 124.41, 129.82,130.40, 136.16 (CF3, Carom), 147.66 (C-6), 151.84 (C-2), 163.09 (C-4).MS-EI: m/z (%) = 366 [M+] (56). Anal. calcd for C18H17F3N2O3: C,59.02; H, 4.68; N, 7.65. Found: C, 59.10; H, 4.66; N, 7.64.

5-Isopropyl-1-(prop-2-ynyloxymethyl)-6-(3-trifluoromethylbenzyl)uracil 11bYield 308 mg (81%) as white solid; mp. 141 –1428C. 1H-NMR(CDCl3): d (ppm) = 1.30 (d, 6H, J = 6.9 Hz, 26CH3), 2.45 (t, 1H, J =2.4 Hz, CH:), 2.81 (hept, 1H, J = 6.9 Hz, CH), 4.25 (s, 2H, CH2),4.29 (d, 2H, J = 2.4 Hz, CH2), 5.20 (s, 2H, CH2), 7.27–7.58 (m, 4H,Harom), 9.57 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 20.31 (CH3),28.48 (CH), 33.29 (CH2), 57.15 (CH2), 72.63 (CH:), 74.90 (CH2),78.88 (:C), 120.44 (C-5), 124.19, 124.23, 124.26, 124.31, 129.76,130.48, 136.31 (CF3, Carom), 147.06 (C-6), 151.90 (C-2), 162.23 (C-4).MS-EI: m/z (%) = 380 [M+] (34). Anal. calcd for C19H19F3N2O3: C,60.00; H, 5.03; N, 7.36. Found: C, 59.95; H, 4.97; N, 7.25.

6-[3,5-Bis(trifluoromethyl)benzyl]-5-ethyl-1-(prop-2-ynyloxymethyl)uracil 11cYield 342 mg (79%); mp. 155 –1568C. 1H-NMR (CDCl3): d (ppm) =1.05 (t, 3H, J = 7.4 Hz, CH3), 2.42 (q, 2H, J = 7.4 Hz, CH2), 2.43 (t, 1H,J = 2.3 Hz, CH:), 4.27 (d, 2H, J = 2.3 Hz, CH2), 4.29 (s, 2H, CH2),7.60 (s, 2H, Harom), 7.83 (s, 1H, Harom), 9.57 (s, 1H, NH). 13C-NMR(CDCl3): d (ppm) = 13.63 (CH3), 19.29 (CH2), 33.19 (CH2), 57.17(CH2), 72.59 (CH:), 75.08 (CH2), 78.67 (:C), 118.04 (C-5), 121.61,124.74, 127.48, 132.91, 137.93 (CF3, Carom), 146.48 (C-6), 151.62(C-2), 162.86 (C-4). HRMS – MALDI: m/z = 457.0957 [M+Na+](C19H16F6N2O3Na+); requires 457.0954. Anal. calcd forC19H16F6N2O3: C, 52.54; H, 3.71; N, 6.45. Found: C, 52.60; H, 3.68;N, 6.38.

5-Ethyl-6-(3-fluorobenzyl)-1-(prop-2-ynyloxymethyl)uracil11eYield 168 mg (66%) as white solid; mp. 109 –1118C. 1H-NMR(CDCl3): d (ppm) = 1.08 (t, 3H, J = 7.5 Hz, CH3), 2.46 (s, 1H, :CH),2.47 (q, 2H, J = 7.5 Hz, CH2), 4.16 (s, 2H, CH2), 4.29 (s, 2H, CH2C:),5.19 (s, 2H, CH2), 6.84 –7.36 (m, 4H, Harom), 9.50 (s, H, NH). 13C-NMR(CDCl3): d (ppm) = 13.76 (CH3), 19.15 (CH2), 33.11 (CH2), 57.21(CH2), 72.52 (:CH), 74.82 (CH2), 78.95 (C:), 114.47 (d, J = 21.0 Hz,Carom), 122.94 (d, J = 3.0 Hz, Carom), 130.83 (d, J = 8.3 Hz, Carom),137.53 (d, J = 7.5 Hz, Carom), 163.26 (d, J = 246.0 Hz, Carom), 117.38(C-5), 148.08 (C-6), 151.85 (C-2), 163.09 (C-4).

6-(2,5-Difluorobenzyl)-5-ethyl-1-(prop-2-ynyloxymethyl)uracil 11fYield 183 mg (73%) as white solid; mp. 108 –1108C. 1H-NMR(CDCl3): d (ppm) = 1.06 (t, 3H, J = 7.5 Hz, CH3), 2.45 (s, 1H, :CH),2.42 (q, 2H, J = 7.5 Hz, CH2), 4.13 (s, 2H, CH2), 4.27 (s, 2H, CH2C:),5.22 (s, 2H, CH2), 6.64 –7.13 (m, 3H, Harom), 9.51 (s, H, NH). 13C-NMR (CDCl3): d (ppm) 13.62 (CH3), 19.12 (CH2), 26.80 (d, J = 3.0 Hz,CH2), 57.15 (CH2), 72.61 (:CH), 74.90 (CH2), 78.69 (C:), 114.47 –117.03, 124.06, 124.16, 124.30, 124.41, 156.38 (d, J = 243.8 Hz,Carom), 158.99 (d, J = 240.00 Hz, Carom), 117.89 (C-5), 147.04 (C-6),151.76 (C-2), 162.89 (C-4). HRMS – MALDI: m/z = 357.1021 [M+Na+](C17H16F2N2O3Na+); requires 357.1016.

5-Ethyl-1-(prop-2-ynyloxymethyl)-6-(2,3,5,6-tetrafluorobenzyl)uracil 11hYield 93 mg (38%). 1H-NMR (CDCl3): d (ppm) = 0.94 (t, 3H, J =7.2 Hz, CH3), 2.41 (s, 1H, :CH), 2.39 (q, 2H, J = 7.2 Hz, CH2), 4.18(s, 2H, CH2), 4.27 (s, 2H, CH2C:), 5.43 (s, 2H, CH2), 6.99-7.15 (m,1H, Harom), 9.11 (s, H, NH). 13C-NMR (CDCl3): d (ppm) = 12.89 (CH3),19.18 (CH2), 23.49 (CH2), 57.17 (CH2), 72.98 (:CH), 74.83 (CH2),78.66 (C:), 105.13–115.75 (Carom), 117.63 (C-5), 146.39 (C-6),151.55 (C-2), 162.66 (C-4). HRMS – MALDI: m/z = 393.0833 [M+Na+](C17H14F4N2O3Na+); requires 393.0837.

5-Ethyl-6-(3-fluorobenzyl)-1-(2-methylallyloxymethyl)uracil 12eYield 219 mg (82%) as white solid; mp. 105 –1078C. 1H-NMR(CDCl3): d (ppm) = 1.07 (t, 3H, J = 7.5 Hz, CH3), 1.71 (s, 3H, CH3),2.45 (q, 2H, J = 7.5 Hz, CH2), 4.02 (s, 2H, CH2), 4.18 (s, 2H, CH2),4.89, 4.95 (26s, 2H, =CH2), 5.14 (s, 2H, CH2), 6.82–7.35 (m, 4H,Harom), 9.53 (s, H, NH). 13C-NMR (CDCl3): d (ppm) = 13.73 (CH3),19.14 (CH2), 19.44 (CH3), 33.10 (CH2), 72.71 (CH2), 73.59 (CH2),112.65 (=CH2), 141.12 (C=), 114.27-114.55 (m, 26Carom), 122.95 (d,J = 3.0 Hz, Carom), 130.80 (d, J = 8.3 Hz, Carom), 137.69 (d, J = 7.5 Hz,Carom), 163.27 (d, J = 246.0 Hz, Carom), 117.22 (C-5), 148.25 (C-6),151.79 (C-2), 163.20 (C-4). HRMS – MALDI: m/z = 355.1434 [M+Na+](C18H21FN2O3Na+); requires 355.1400. Anal. calcd for C18H21FN2O3:C, 65.05; H, 6.37; N, 8.43. Found: C, 65.22; H, 6.37; N, 8.32.

6-(2,5-Difluorobenzyl)-5-ethyl-1-(2-methylallyloxymethyl)uracil 12fYield 190 mg (72%) as white solid; mp. 96–998C. 1H-NMR (CDCl3):d (ppm) = 1.05 (t, 3H, J = 7.5 Hz, CH3), 1.70 (s, 3H, CH3), 2.41 (q, 2H,J = 7,5 Hz, CH2), 4.00 (s, 2H, CH2C=), 4.14 (s, 2H, CH2), 4.87, 4.94(26s, 2H, =CH2), 5.15 (s, 2H, CH2), 6.62–7,12 (m, 3H, Harom), 9.44 (s,H, NH). 13C-NMR (CDCl3): d (ppm) = 13.60 (CH3), 19.12 (CH3), 19.38(CH2), 26.70 (d, J = 3.0 Hz, CH2), 72.83 (CH2), 73.54 (CH2), 112.67(=CH2), 140.99 (C=), 114.48–117.01, 124.19 –124.52, 156.37 (d, J =243.8 Hz, Carom), 158.98 (d, J = 243.00 Hz, Carom), 117.69 (C-5),147.25 (C-6), 151.63 (C-2), 162.95 (C-4). HRMS – MALDI: m/z =373.1334 [M+Na+] (C18H20F2N2O3Na+); requires 373.1331. Anal.calcd for C18H20F2N2O3: C, 61.71; H, 5.75; N, 8.00. Found: C, 61.77;H, 5.62; N, 7.92.

5-Ethyl-1-(2-methylallyloxymethyl)-6-(2,3,5,6-tetrafluorobenzyl)uracil 12hYield 144 mg (56%).1H-NMR (CDCl3): d (ppm) = 1.04 (t, 3H, J =7.5 Hz, CH3), 2.49 (q, 2H, J = 7.5 Hz, CH2), 4.15 (m, 2H, CH2), 4.20 (s,2H, CH2), 5.41 (s, 2H, CH2), 5.14–5.34 (m, 2H, =CH2), 5.76 –5.95(m, 1H, CH=), 7.00 –7.15 (m, 1H, Harom), 9.00 (s, H, NH). 13C-NMR(CDCl3): d (ppm) = 13.22 (CH3), 18.77 (CH2), 24.03 (CH2), 69.92(CH2), 71.31 (CH2), 117.85 (=CH2), 133.25 (CH=), 105.02–106.09,117.21 –117.45 (Carom), 114.14 (C-5), 146.56 (C-6), 151.45 (C-2),162.75 (C-4).

HRMS – MALDI: m/z = 395.0989 [M+Na+] (C18H18F4N2O3Na+);requires 395.0971.

Antiviral assay proceduresCompounds were solubilized in DMSO at 100 mM and thendiluted in culture medium. Cells and viruses: MT-4, C8166, andH9/IIIB cells were grown at 378C in a 5% CO2 atmosphere in RPMI1640 medium supplemented with 10% fetal calf serum (FCS),100 IU/mL penicillin G, and 100 lg/mL streptomycin. Cell cul-



tures were checked periodically for the absence of mycoplasmacontamination with a MycoTect Kit (Gibco). Human immunode-ficiency viruses type 1 (HIV-1, IIIB strain) was obtained fromsupernatants of persistently infected H9/IIIB cells. The HIV-1stock solutions had titers of 4.56106, 50% cell culture infectiousdose (CCID50)/mL. The K103R + V179D + P225H mutant (EFVR) wasderived from an IIIB strain passaged in MT-4 cells in the presenceof efavirenz (up to 2 lM). The Y181C mutant (NIH N119) wasderived from an AZT-sensitive clinical isolate passaged initiallyin CEM and then in MT-4 cells in the presence of nevirapine(10 lM). The double mutant K103N + Y181C (NIH A17) wasderived from the IIIB strain passaged in H9 cells in the presenceof BI-RG 587 (1 lM). EFVR, N119, and N117 stock solutions hadtiters of 4.06105, 1.26108, and 2.16107 CCID50/mL, respectively.HIV titration: Titration of HIV was performed in C8166 cells bythe standard limiting dilution method (dilution 1 : 2, four rep-lica wells per dilution) in 96-well plates. The infectious virus titerwas determined by light microscope scoring of syncytia after4 days of incubation. Virus titers were expressed as CCID50/mL.

Anti-HIV assaysThe activity of test compounds against multiplication of wildtype HIV-1, EFVR, N119, and N117 in acutely infected cells wasbased on inhibition of virus-induced cytopathicity in MT-4 cells.Briefly, an amount of 50 lL of culture medium containing26104 cells was added to each well of flat-bottom microtitertrays containing 50 lL of culture medium with or without vari-ous concentrations of test compounds. Then, an amount of20 lL of HIV suspensions (containing the appropriate amount ofCCID50 to cause complete cytopathicity at day 4) was added. Afterincubation at 378C, cell viability was determined by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT)method [31]. The cytotoxicity of test compounds was evaluatedin parallel with their antiviral activity and was based on the via-bility of mock-infected cells, as monitored by the MTT method.

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Synthesis and Antiviral Evaluation of 3′Branched Nucleoside Analogs

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