5504
`
`J. Med. Chem. 2005, 48, 5504-5508
`
`Design, Synthesis, and Antiviral Activity of
`2'-Deoxy-2'-fluoro-2'-C-methylcytidine, a Potent Inhibitor of Hepatitis C Virus
`Replication
`
`Jeremy L. Clark,+,-;, Laurent Hollecker,+,® J. Christian Mason/·* Lieven J. Stuyver,t,II Phillip M. Tharnish,t
`Stefania Lostia,t·® Tamara R. McBrayer,t Raymond F. Schinazi,t Kyoichi A. Watanabe,*,t Michael J. Otto,t
`Phillip A. Furman,t Wojciech J. Stec,t,.1 Steven E. Patterson/·# and Krzysztof W . Pankiewiczt.#
`
`Pharmasset, Inc., 303-A College Road East, Princeton, New ,Jersey 08540, and Department of Pediatrics, Emory University,
`School of Medicine/Veterans Affairs Medical Center, Decatur, Georgia 30033
`
`Received March 28, 2005
`
`The pyrimidine nucleoside beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a
`hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound
`was obtained by a DAST fluorination of N4-benzoyl-l-(2-methyl-3,5-di-O-benzoyl-/3-o-arabino(cid:173)
`furanosyl]cytosine (6) to provide N4-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-/3-D-ribofura(cid:173)
`nosyl]cytosine (7a). The protected 2'-C-methylcytidine (7c) was obtained as a byproduct from
`the DAST fluorination and allowed for the preparation of two biologically active compounds
`from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic
`HCV replicon assay system and found to be potent and selective inhibitors ofHCV replication.
`Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'(cid:173)
`C-methylcytidine and low cellular toxicity.
`
`Introduction
`Hepatitis C virus infection is a major health problem
`that leads to chronic liver disease, such as cirrhosis and
`hepatocellular carcinoma, in a substantial number of
`infected individuals. Once infected, about 20% of people
`clear the virus, but the rest can harbor HCV the rest of
`their lives. Ten to 20% of chronically infected individuals
`eventually develop liver-destroying cirrhosis or cancer.
`The current standard of care for chronic hepatitis C is
`combination therapy with an interferon-a. and ribavirin.
`Studies have shown that more patients with hepatitis
`C respond to pegylated interferon-a/ribavirin combina(cid:173)
`tion therapy than to combination therapy with unpegy(cid:173)
`lated interferon-a.. The overall response rate to treat(cid:173)
`ment, defined as loss of HCV from serum 6 months after
`completion of treatment, is 40%. Because of the low
`response rates as well as toxic side effects and unsus(cid:173)
`tained viral load reductions, these therapies are inad(cid:173)
`equate. Moreover, there is no established vaccine for
`HCV, and there is an urgent need for improved thera(cid:173)
`peutic agents that effectively combat chronic HCV
`infection. 1
`The nonstructural protein NS5B has been character(cid:173)
`ized as an RNA-dependent RNA polymerase (RdRp) that
`is required for viral replication. This polymerase is
`considered to be an essential component in the HCV
`
`* Corresponding author. Tel: +1-619-613-4100. Fax: +l-619-613-
`4150. E-mail: kwantanabe@11harmasset.com.
`' Pharmasset, Inc.
`' Emory University.
`~ Current address: Southern Research lnstitute, 2000 Ninth Ave.
`South, Birmingham, AL.
`'"Current address: Via San F ili ppo, 19, Monserrato (CA), 09042,
`Italia.
`1 Deceased April 2005.
`11 Current address: Vireo BVBA, Mechelen, Belgium.
`~ Current address: Centre of Molecular and Macromolecu lar Stud(cid:173)
`ies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 L6dz, Poland.
`• Current address: UMN Center for Drug Design, 7- 146, Phillips(cid:173)
`Wangensteen Bldg., 516 Delaware St SE, Minneapolis, MN 55455.
`
`2'-C-CH3..:ytidine
`2'-FdCyd
`Figure 1. Structures of 2' -deoxy-2' -fluoro-2' -C-methylcytidine
`(1), 2'-C-methylcytidine, and 2'-deoxy-2'-fluorocytidine (2'(cid:173)
`FdCyd).
`
`replication complex and therefore is an ideal target for
`drug discovery. Recently, several 2'-modified nucleoside
`analogues with potent inhibitory activity against the
`HCV NS5B polymerase have been identified. Among the
`most potent compounds in this class are 2'-deoxy-2'(cid:173)
`fluorocytidine (2'-FdCyd)2 and 2'-C-methyl nucl eosides
`(Figure 1)_3 - 5 Despite the potent HCV inhibition of 2'(cid:173)
`FdCyd, its thera peutic potential as an antiviral agent
`is diminished due to a lack of selectivity between host
`cells and the viral target. 2'-FdCyd triphosphate has
`been demonstrated to be a substrate for both RNA and
`DNA polymerases.6-7 Here we describe the synthesis and
`biological activity of 2'-deoxy-2'fluoro-2'-C-methyl cyti(cid:173)
`dine (1 ) as a potent anti-HCV agent.
`
`Chemistry
`For the synthesis of2'-deoxy-2'-fluoro-2'-C-methylcy(cid:173)
`tidine (1 ), N 4-benzoyl-1-(2-methyl-3 ,5-di-O-benzoyl-j'i-o(cid:173)
`arabinofuranosyllcytosine (6) was chosen as the key
`intermediate and was prepared in approximately 20%
`yield in six steps from cytidine (Scheme 1).8 Briefly,
`selective benzoylation of cytidine with benzoic anhydride
`in DMF,9 followed by treatment with TIDPSCiz in
`pyridine, afforded N 4-benzoyl-3' ,5' -0-( tetraisopropyl(cid:173)
`disiloxane-1,3-diyl)cytidine (2). 10 Oxidation of the 2'(cid:173)
`alcohol to the 2'-ketone derivative (3) was achieved with
`
`10.1021/jm0502788 CCC: $30.25 © 2005 American Chemical Society
`Published on Web 07/26/2005
`
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`2'-Deoxy-2'-fiuoro-2'-C-methylcytidine
`
`Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5505
`
`Scheme 1°
`
`;riz
`lNAO
`ROt,,--o_j
`)----f-OH
`RO
`CH3
`d~ 4: R=TIDPS
`:.· R = H
`,--
`~ R = Bz
`a Reagents and conditions: (a) (i) Bz2O, DMF, room temp, (ii) TIDPSCh, DMF; (b) DMSO, TFAA, TEA, -15 °C; (c) MeLi, -78 °C; (d)
`1 M TBAF, coned HOAc, rt; (e) BzCl, pyridine, rt.
`
`;r:z
`lNAO
`r1dr~ ~
`
`0
`
`0
`
`0
`
`65
`
`8
`
`Cytldlne
`
`3
`
`7a
`
`8
`
`9
`
`0 100.2, d, 1J c- F = 187. 7
`
`0 99.9, d, 1J c- F = 186.2
`() 102.1, d, 1J c - F = 180.1
`
`Scheme 2°
`
`NHBz
`
`CN
`
`B,Ow~O
`
`O
`
`a
`OH-->-
`
`BzO
`
`CH3
`
`6
`
`NHR
`
`CN
`NAO
`
`RO
`
`~CH3
`RO
`F
`
`NHR
`
`+ RO\=i
`
`CN
`NAO
`
`+
`
`RO
`
`CH2
`
`NHR
`
`CN
`NAO
`
`RO
`
`~CH3
`RO
`OH
`
`7c· R = Bz
`b ~2'-MeC, R = H
`
`7a· R = Bz
`7b: R = Bz
`b~1;R=H
`b~DMDC,R=H
`a Reagents and conditions: (a) DAST, toluene, -20 °C to rt; (b) MeOH/NH3, rt.
`Table 1. Flourine-Coupled 1H and 13C NMR Chemical Shifts, Multiplicities, and J Values for Compounds 1, 7a, 8, and 9"
`H-1/C-1
`C-2
`H-3/C-3
`compd
`2CH3
`0 1.17, d, 3JH-F = 22.3
`0 6.07, d, 3JH-F = 18.9
`1
`0 101.2, d, 1Jc-F = 180.1
`overlapping mult.
`6 16.6, d, 2Jc- F = 25.9
`6 88.6, d, 2Jc-F = 37.4
`0 70.5, d, 2Jc-F = 18.3
`0 1.49, d, 3JH- F = 22.4
`0 6.52, d, 3JH-F = 18.0
`6 5.56, dd, 3JH-F = 20.7
`0 72.4, d, 2Jc- F = 16.0
`0 17.4, d, 2Jc- F = 25.2
`0 91.2, d, 2Jc-F = 42.0
`6 6.17, d, 3JH-F=19.3
`0 1.39, d, 3Jtt - F = 22.3
`6 5.49, d, 3JH - F = 21.2
`() 72 .7, d, 2Jc- F = 16.1
`617.3, d, 2J c- F= 25.1
`6 90.7, d, 2Jc-F=44.2
`6 6.13, d, 3JH-F = 18.9
`6 1.35, d, 3JH- f = 22.3
`overlapping mult.
`6 16.9, d, 2J c- F = 25.2
`6 90.6, d, 2J c-F = 44 .0
`0 72.5, d, 2Jc- F = 17.6
`0 NMR spectra were recorded at 30 °C (400 MHz) in DMSO-d5 for compound 1, CDCl3 for compound 7a, CD 3OD + CDCl3 for 8, and
`CD3OD for 9 with concentrations of -40 mg/0.75 mL. J values are in Hz.
`Upon treating 6 with DAST in toluene or dichloro(cid:173)
`trifluoroacetic anhydride/DMSO under Swem oxidation
`conditions.11 Purification of compound 3 by silica gel
`methane, a clean mixture of three products (7a-7cJ in
`chromatography followed by crystallization from petro(cid:173)
`15-20% yield for each compound was obtained (Scheme
`leum ether-CH2Clz provided a white solid that was
`2). The desired transformation of 6 to N 4-benzoyl-1-l2-
`stable when stored at room temperature with minimal
`fluoro-2-methyl-3,5-di-O-benzoyl]cytosine (7a) proceeded
`atmospheric exposure. 12 Treatment of the 2'-ketone (3)
`with inversion of configuration and was stereospecific;
`with methyllithium at -78 °C in diethyl ether gave
`no diastereomeric N 4-benzoyl-1-[2-fluoro-2-methyl-3,5-
`exclusively the protected 1-[2-C-methyl-3,5-O-(tetrai(cid:173)
`di-O-benzoyl-/3-D-arabinofuranosy1Jcytosine was detected
`sopropyldisiloxane-1 , 3-diyl)-/1-D-arabi nof'uranosyl J cy(cid:173)
`in the crude reaction mixture. The presence of the
`tosine (4). 13 The 3',5'-silyl protecting group was removed
`tertiary fluorine at the 2' position in 7a was confirmed
`with TBAF/acetic acid and replaced with benzoyl pro(cid:173)
`by the 1H and 13C NMR multiplicities and coupling
`tecting groups to provide compound 6.
`constants (Table 1), whereas the stereochemistry of the
`The fluorination of tertiary alcohols using DAST has
`fluorination was determined by nuclear Overhauser
`been reported, but the stereochemistry of such trans(cid:173)
`enhancement 1H NMR difference spectroscopy (Figure
`formations is substrate-specific and often unpredictable.
`2J.
`For instance, Yang et al. reported that the DAST
`Analysis of the 1H NMR spectrum of compound 7a
`fluorination of a tertiary alcohol in 2-bromomethyl-DL(cid:173)
`revealed three distinct multiplicities due to H - F cou(cid:173)
`myo-inositol proceeds with retention of configuration. 14
`pling: a doublet at o 1.49 (2'-CH3), a doublet of doublets
`Wachtmeister et al. obtained a 4-fluoro-1-cyclopentanol
`at o 5.56 (H-3'), and a doublet at o 6.52 (H-1'). Irradia(cid:173)
`containing a tertiary fluorine in 25% yield using DAST
`tion of the H-3' resonance resulted in a relatively large
`as a fluorinating reagent, and this transformation
`enhancement of both the H-5' signal (4.8%) and the 2'(cid:173)
`proceeded with inversion of configuration.15 Further(cid:173)
`methyl signal (5 .9%), while irradiation of the 2'-methyl
`more, dehydrations or eliminations, rearrangements,
`signal resulted in an enhancement of both H-3' !3.4%)
`and ring contractions are often pervading problems in
`the DAST fluorination of highly functionalized mol(cid:173)
`and, to a lesser extent, H-1' (1.7%). Deprotection of7a
`ecules.16
`using methanolic ammonia provided the title compound,
`
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`

`5506 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
`
`Clark et al.
`
`c1::
`
`BzO,CH, 3.4~ NAO
`
`4.8%~1.7%
`
`~~
`
`BzO
`
`F
`
`7a
`Figure 2. 1H NMR NOE correlations of compound 7a.
`
`Figure 3. ORTEP drawing of2'-deoxy-2'-fluoro-2'-C-methyl(cid:173)
`cytidine (1).
`
`Scheme 3"
`
`NHBz C1
`0J O
`
`Bzol-
`
`RO
`
`0
`
`(;:
`
`N
`
`0
`
`~CH3
`F
`BzO
`
`\=?-CH3
`RO
`F
`
`7a
`
`8: R= Bz
`b 1
`~9: R=H
`a Reagents and conditions: (a) 80% HOAc, reflux; (b) MeOH/
`NHa, rt.
`
`1, whose structure was unambiguously confirmed by
`X-ray crystallography and revealed the expected 3'-endo
`conformation (Figure 3).
`The degradation enzymes cytidine deaminase (CDA)
`and deoxycytidine monophosphate deaminase (dCMP(cid:173)
`DA) are responsible for the in vivo metabolic conversion
`of cytidine or cytidine monophosphate to uridine. To
`facilitate future in vivo studies of compound 1, 2'-deoxy-
`2'-fluoro-2'-C-methyluridine (9) was prepared from 7a
`by deamination in refluxing 80% acetic acid followed by
`debenzoylation using methanolic ammonia (Scheme 3).
`
`Results and Discussion
`The novel pyrimidine nucleoside analogues 2'-deoxy-
`2'-fluoro-2'-C-methylcytidine (1) and 2'-deoxy-2'-fluoro-
`2'-C-methyluridine (9) were tested for anti-HCV activity
`in both a cell-based quantitative real-time RT-PCR
`assay and surrogate bovine viral diarrhea virus (BVDV)
`assays as previously described (Table 2). 17 The activity
`and cytotoxicity profiles of 2'-C-methylcytidine and 2'(cid:173)
`deoxy-2'-fluorocytidine (2'-FdCyd) are included for com-
`
`Table 2. Anti-HCV Activity and Cellular Toxicity of
`Compounds 1, 9, 2'-C-Methylcytidine (2'-C-MeCyd), and
`2' -Deoxy-2' -fluorocytidine (2' -FdCyd)
`HCV repliconb
`cpBVDV0 (MDBK cells)
`EC90 (µM)b
`compound
`CC50 (µM) EC90 (µM) CCso' (µM)
`5.40 ± 2.6
`>100
`>100
`> 100
`1
`>100
`>100
`> 100
`> 100
`9
`·2.30 ± 0.1
`19.0 ± 5.7
`>100
`> 100
`2-C-MeCyd
`6.50 ± 1.6
`>100
`>100
`2-FdCyd
`>100
`a cpBVDV = cytopathic BVDV. b 96 h, average of at least four
`experiments. ' MTS CCso was determined in a 4-day assay using
`the Celltiter 96 nonradioactive cell proliferation assay from
`Promega (Madison, WI).
`
`parison and indicate that compound 1 demonstrated a
`similar potency as 2'-FdCyd in the HCV replicon assay.
`Dynamic profiling of the cell growth in this replicon
`assay revealed no cytostasis for compound 1 at the HCV
`replicon EC90 value. As previously reported, 2'-FdCyd,
`although not cytotoxic, induced cytostasis at the EC90
`value. 2 Additionally, much like 2'-FdCyd, but unlike 2'(cid:173)
`C-MeCyd, compound 1 was inactive in the BVDV
`assays. Compound 9 demonstrated no activity or
`cytoxicity in any assay.
`
`Experimental Section
`All reagents and anhydrous solvents were purchased from
`Aldrich or Acros and were used as received. 1H, 19F, and 13C
`NMR spectra were obtained with a Varian Unity Plus 400
`spectrometer at 400, 376, and 100 MHz, respectively. 1H and
`uc NMR chemical shifts are reported as o (ppm) downfield
`with respect to an internal standard of tetramethylsilane,
`while 19F chemical shifts are reported downfield from an
`external standard ofhexafluorohenzene. Optical rotations were
`measured with a Perkin-Elmer 241 automatic polarimeter at
`the sodium D line (589 nm) in a 1-dm cell. Melting points were
`determined using an electrothermal digital melting point
`apparatus and are uncorrected. Atlantic Microlab, Inc. of
`Norcross, GA provided the elemental analysis.
`N 4-Benzoyl-1-[2-C-methyl-3,5-O-(tetraisopropyldisilox(cid:173)
`ane-l,3-diyl)-p-o-arabinofuranosyl]eytosine (4). Com(cid:173)
`pound 3 (37.6 g, 64 mmol) was dissolved in anhydrous Et20
`(800 mL) under argon and cooled to - 78 °C with stirring. To
`this solution was added MeLi (103 mL, 1.6 Min Et20) dropwise
`over 1 h. After stirring for an additional 2 h, the reaction
`mixture was quenched by dropwise addition of 1 M NH. Cl (165
`mL). Upon warming to room temperature, the mixture was
`diluted with EtOAc (600 mL) and H 20 (130 mL). The orgru,ic
`phase was separated, washed with H20 (1 x 130 mL), dried
`(Na2S04), and the concentrated to dryness to give a brown
`foam (42.5 g, > 100%) that was used without further purifica(cid:173)
`tion. An analytical sample was obtained by silica gel chroma(cid:173)
`tography eluting with 2: 1 Et20-petroleum ether: [<l!2°r, +52.2°
`(c 1, CHCh); 1H NMR (CDCbJ o 0.96- 1.15 (m, 24H), 1.60 ls,
`3Hl, 3.81 (dt, lH, J = 1.9, 9.2 Hz), 4.02 (dd, lH, J = 2.5, 13.7
`Hz), 4.17 - 4.23 (m, 2H), 5.85 (s, lH), 7.50-7.54 (m, 2H), 7.60-
`7.64 (m, 2H), 7.91 ld, 2H, J = 7.3 Hz), 8.38 (d, lH, J = 7.3
`Hz), 8.89 (bs, lH); 13C NMR (CDC!a) ,l 12.5, 13.0, 13.1, 13.6,
`16.9, 17.1, 17.2, 17.4, 17.5, 17.6, 17.7, 20.9, 60.4, 72.9, 81.7,
`91.2, 96.5, 127.8, 129.0, 133.2, 145.2, 156.6, 162.5, 166.7. Anal.
`Calcd (C29H4sN301Si2): C, 57.68; H, 7.51; N, 6.96. Found: C,
`57.63; H, 7.55; N, 6.82.
`N 4-Benzoy l-2 '-C-methy 1-P-n-ara bi no fur an osyl cyt i(cid:173)
`dine (5). Crude 4 (128.0 g, 0.212 moll was dissolved in THF
`(1.28 L) and treated with glacial HOAc (23.0 mL, 0.401 mol).
`To this solution was added tetrabutylammonium fluoride (384
`mL, 1 Min THF) at room temperature a nd stirred for 0.75 h.
`The mixture was treated with silica gel (750 gJ and concen(cid:173)
`trated to dryness in vacuo, and the tan-colored residue was
`placed onto a silica gel column. Eluting with 1:7 EtOH - CHr
`Clz afforded a waxy solid that was preadsorbed onto silica gel
`
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`2'-Deoxy-2'-fluoro-2'-C-methylcytidine
`
`Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5507
`
`(300 g) and chromatographed as before to give an off-white
`solid (46.4 g, 61 %). Crystallization from aqueous acetone
`afforded an analytical sample: mp 197- 200 °C; [al22n + 132.0°
`(c l, MeOH); 1H NMR (DMSO-d6) cl 1.20 (s, 3H), 3.62-3.69
`(m, 2H), 3.73-3.78 (m, 2H), 5.19 (t, lH, J = 5.4 Hz), 5.25 (s,
`lH), 5.52 (d, lH, J = 5.0 Hz), 5.99 (s, lHJ, 7.32 (d, lH, J = 5.8
`Hz), 7.50 (Wt, 2H, J = 7.7 HzJ, 7.62 (Wt, lH, J = 7.3 Hz), 8.00
`(d, 2H, J = 7.3 Hz), 8.14 (d, lH, J = 6.9 Hz), 11.22 (s, lH); tjC
`NMR (DMSO-dGl o 19.6, 61.3, 77.5, 78.5, 85.2, 88.9, 95.2, 128.5,
`132.8, 133.3, 147.3, 154.9, 162.9, 167.4. Anal. Calcd (C11H1gN,1O6 •
`0.5H2O): C, 55.14; H , 5.41; N, 11.35. Found: C, 55.21; H, 5.47;
`N, 11.33.
`N 4-Benzoyl-1-(2-C-methyl-3,5-di-O-benzoyl-P-D-arabino(cid:173)
`furanosyl]cytosine (6). Compound 5 (46.0 g, 0.127 mo)) was
`dissolved in anhydrous pyridine (200 mL) and the solvent was
`removed in vacuo. The resulting syrup was dissolved in
`anhydrous pyridine, cooled to O °C under argon with stirring,
`and treated with BzCl (30.0 mL, 0.250 mo]) dropwise over 10
`min. After the addition was complete, the ice bath was
`removed and stirring was continued for 1.5 h. Water (5 mL)
`was added and the mixture was concentrated to dryness in
`vacuo. The residue was dissolved in CH2Cb and washed with
`saturated NaHCO3 (1 x 500 mL) and water (1 x 500 mL).
`The organic layer was dried (Na2SO4), filtered, and concen(cid:173)
`trated to dryness to afford a syrup that was purified by silica
`gel chromatography eluting with 1:1 EtOAc-hexanes. Com(cid:173)
`pound 6 was isolated as an off-white solid (48.5 g, 67%): [al2 2n
`+ 9.6° (c 1, CHC!a); 1H NMR (CDCU 1) 1.64 (s, 3H), 4.50 (m,
`lH), 4.78-4.85 (m, 2H), 5.50 (d, lH, J = 3.4 Hz), 6.42 (s, lH),
`7.44-7.54 (m, 7H), 7.57-7.66 (m, 3H), 7.94 (d, 2H, J = 7.8
`Hz), 8.05-8.09 (m, 4H), 8.21 (d, lH, J = 7.3 Hz); 13C NMR
`(CDCb) o 19.9, 64.4, 79.2, 80.6, 81.6, 90.3, 96.7, 128.1, 128.6,
`128.8, 128.9, 129.5, 129.9, 130.0, 133.2, 133.5, 134.0, 14 7.5,
`156.0, 162.7, 165.9, 166.6. Anal. Calcd (C31H 2, N3OgJ: C, 65.37;
`H , 4 .78; N, 7.38. Found: C, 65.59; H , 4.79; N, 7.16.
`Reaction of 6 with DAST. To a stirred solution of 6 (2.96
`g, 5.20 mmol) in anhydrous toluene (50 mL) was added DAST
`(1.0 mL, 7.8 mmol) at -20 °C under argon. After the addition
`was complete, the cooling bath was removed and stirring was
`continued for 1 h. The reaction mixture was poured into
`saturated NaHCO3 (50 mL) and washed until gas evolution
`ceased. The organic phase was dried (Na2SO.), concentrated
`to dryness, and purified by silica gel chromatography eluting
`with 1:1:1 EtOAc- CHCb-hexanes to afford compound 7a
`(0.55 g, 19%) as a white solid, followed by compound 7b (0.39
`g, 14%) as an off-white solid. Elution was continued with 1:1:1
`EtOH-EtOAc-CHCl.i-hexanes to afford compound 7c (0.451
`g, 15%) as an off-white solid. Analytical samples were obtained
`by recrystallization from the indicated solvents.
`N 4 -Benzoyl-3',5'-di-O-benzoyl-2'-fluoro-2'-C-methyl(cid:173)
`cytidine (7a): mp 241 °C (CH 2C[s-hexanes); laF 2n +82.0° (c
`1, CHCh); 1H NMR (CDCb) o 1.49 (d, 3H, J = 22.4 Hz), 4.64
`(dd, lH, J = 3.44, 12.9 Hz), 4.73 (d, lH , J = 9.5 Hz), 4.90 /dd,
`lH, J = 2.4, 12.7 Hz), 5.56 (dd, lH, J = 8.6, 20.7 Hz), 6.52 (d,
`lH, J = 18.0 Hz), 7.47-7.57 (m, 7H), 7.62-7.71 (m, 3H), 7.89
`(d, 2H, J = 6.9 Hz), 8.07-8.11 (m , SH), 8.67 (bs, lH); 13C NMR
`(CDCbJ o 17.4 (d, J = 25.2 Hz), 62.1 , 72.4 (d, J = 16.0 Hz),
`77.7, 91.2 (d, J = 42.0 Hz), 97.5, 100.2 (d, J = 187.7 Hz), 127.8,
`128.6, 128.8, 128.9, 129.2, 129.6, 129.7, 130.3, 133.2, 133.4,
`133.8, 134.1, 143.8, 154.6, 162.6, 165.6, 166.1; 19F NMR (CDCb)
`rl 3.9 Im ). Anal. Calcd (C31H2oFN,1Or 0.7H2O1: C, 63.74; H , 4.73;
`N, 7.19. Found: C, 63.71; H, 4.54; N, 7.20.
`N 4-Benzoyl-1-l2-dcoxy-2-methylidenc-3,5-di-O-benzoyl(cid:173)
`/J-D-glycero-pentofuranosyl]cytosine (7b): mp 173.4 - 174.4
`0 -40.4° (c l, CHC!a); 1H NMR (CDC13): cl 4.58
`(EtOH); [a]22
`(dd, lH, J= 3.7, 5.0 Hz), 4.70-4.81 (m, 2H), 5.55 (s, HI), 6.09-
`6.11 (m, lH), 7.03 (d, lH, J = 1.3 Hz), 7.40-7.66 (m, l0H),
`7.85 (d, lH, J = 7.3 Hz), 7.91 (d, 2H, J = 7.7 Hz), 8.03 (dd,
`2H, J = 0.9, 8.3 Hz), 8.03 (dd, 2H), 8.86 (bs, lH); 13C NMR
`(CDCl3) o 63.9, 73.3, 80.7, 85.9, 97.8, 117.3, 127.8, 128.0, 128.7,
`128.9, 129.1, 129.4, 129.6, 129.9, 130.2, 133.0, 133.6, 133.8,
`144.1, 144.9, 155.1, 162.5, 165.9, 166.1. Anal. Calcd (C31H 25-
`N3O1): C, 67.51; H, 4.57; N, 7.62. Found: C, 67.21; H, 4.51;
`N, 7.66
`
`N 4 -Benzoyl-2' -C-methyl-3' ,5' -di-0-benzoy lcytidine
`(7c): mp 176.7-179.1 °C (EtOH); [a]22
`0 +46.2° (c 1, CHCla);
`1H NMR (CDC1 3) ,I 1.29 (s , 3H), 4.67-4.80 (m, 3Hl, 4.82-4.87
`(m, lH), 5.30 (d, lH, J = 5.8 Hz), 6.09 (s, lH), 7.47-7.56 (m,
`4H), 7.89 (d, 2H, J == 7.3 Hz), 8.07-8.14 (m, 4Hl, 8.68 (s, lH);
`i:JC NMR (CDC!:;) o 21.4, 62.7, 75.8, 78.5, 79.0, 93.4, 97.3, 127.8,
`128.5, 128.7, 128.9, 129.0, 129.4, 129.6, 130.0, 132.9, 133.2,
`13:3 .6, 144.3, 156.0, 16Z.8, 165.8 166.2. 166.8. Anal. Cakd
`(C31H21N JO8·0.4H2Ol: C, 64.56; H, 4.86; N , 7.29. Found : C,
`64.54; H, 4.81; N, 7. 32.
`3' ,5' -Di-0-benzoyl-2' -deoxy-2' -fl uoro-2' -methyl uri(cid:173)
`dine (8). Compound 7a (0.225 g, 0.394 mmol) was suspended
`in 80% aqueous HOAc ( 15 mL) and heated under reflux with
`stirring for 12 h. The clear solution was cooled, concentrated
`to dryness in vacuo, and coevaporated with 50% MeOH -water
`(3 x 5 mL) to remove the residual HOAc. Purification by silica
`gel chromatography, eluting with 2% EtOH-CH2C[z, gave
`0.160 g of 8 (87%) as a white solid. Crystallization from
`2-propanol afforded an analytical sample: mp 256.4- 257.6 °C;
`[aFio + 71.7° (c 1, CHCh); 1H NMR (CDCh + CD3ODJ r\ 1.39
`(d, 3H, J = 22.3 Hz), 4.49 (dd, lH, J = 3.9, 12.7 Hz), 4.57 (m,
`UI,), 4 .79 (dd, HI, J = 2.7, 12.5 H zl, 5.42 (d, HI, J = 8.1 Hz J,
`5.49 !<l<l, lH, J = 9.20, 21.2 Hz). 6.17 (d, llI, J = 19.3), 7.37
`7.50 (m , 4H), 7.51-7.57 (m, 3H, H-6) 7.93-8.01 (m, 4H); uc
`NMR (CDCb + CDaOD) o 17.3 (d, J = 25.1 Hz), 62.1, 72.7 (d,
`J = 16.1 Hz), 90.7 (d, ,J = 44.2 Hz), 99.9 (d, J = 186.2 Hz),
`103.1, 128.5, 128.6, 128.7, 129.4, 129.5, 130.1, 133.6, 134.0,
`139.3, 150.4, 163.2, 165.7, 166.1; 19F NMR (CDCh + CD3OD)
`cl 6.02 (m). Anal. Calcd (C24H 21FN2O1): C, 61.54; H, 4.52; N,
`5.98. Found: C, 61.4~; H, 4.51; N, 5.96.
`General Procedure for Deprotection. The free nucleo(cid:173)
`sides were prepared by treating compounds 7a-c and 8 with
`NH3/MeOH (ca. 7 N, ~ 12 mUmmol) followed by stirring at
`room temperature overnight (8-12 h). The solvent was
`removed in vacuo, a nd the compounds were isolated as
`indi cated
`2'-Deoxy-2'-f1uoro-2' -C-methylcytidine (1 l. Compound
`7a (6.30 g, 0.011 molJ was deprotectecl to give 1 (2.18 g, 76'!o)
`as a white powder after column chromatography eluting with
`9% EtOH in CHCh and then 17% EtOH and finally 25% EtOH
`in CHCb: mp 216.4-218.0 °C (EtOHJ; [c1F2o + 125.6° (c 1,
`H2O); 1H NMR (DMSO-d6) a 1.17 (d, 3H, J = 22.3 HzJ, 3.63
`(dd, lH, J = 2.7, 13.7 Hz), 3.70-3.84 (m, 3H), 5.24 (apµ s,
`lH), 5.60 (d, lH, J = 5.4 Hz), 5.74 (d, lH, J = 7.71 Hz), 6.07
`(d, lH, J = 18.9 Hz), 7.31 (s, lH, NH2l, 7.42 (s, lH, NH2J, 7.90
`(d, lH, J = 7.3 Hz); 13C NMR (DMSO-d 6) n 16.6 (d, J = 25.9
`Hz), 58.5, 70.5 (d, J = 18.3 Hz), 81.4, 88.6 (d, J = 37.4 Hz),
`94.4, 101.2 (d, J = 180.1 Hz), 140.5, 154.8, 165.2; rnF NMR
`(DMSO-d 6 ) a 2.60 (ml. Anal. Calcd (C1oH 14FN3O.·1.5H20l: C,
`41.96; H , 5.94; N, 14.69. Found: C, 42. 24; H , 5.6:3; N, 14.5-l
`Compound 1 was converted to the HCl salt and crystallized
`from aqueous ethanol: mp 243 °C (dee); [aJ 22n + 108.4° (c 1,
`H 2O); 1H NMR (DMSO-d6 ) n 1.29 (d, 3H, J = 22.6 Hz), 3.65
`(dd, lH, J = 2.3, 12.7 Hz), 3.76-3.90 (m, 3H), 5.96 (d, lH, J
`= 17.3 Hz), 6.15 (d, lH, J = 7.9 Hz), 8.33 (d, lH, J = 7.9 H z),
`8.69 (s, 1.5H), 9.78 (s, 1.5Hl; 1:ic NMR (DMSO-d 6 ) n 16.2 (d, J
`= 24.4 Hz), 58.2, 69.9 (d, J = 16.8 Hz), 82 .1, 88.8 (d, J = 32.0
`Hzl, 94.6, 101.1 (d, J = 181.5 Hz), 143.2, 147.6, 159.6; '~F NMR
`(DMSO-d6 ) 6 l.69 (m). Anal. Calcd (C10H15CJFN3O.): C, 40.62;
`H, 5.11; N, 14.21. Found: C, 40.80; H, 5.09; N, 14.23.
`2' -Deoxy-2' -fluoro-2' -C-methyluridine (9). Deprotection
`of8 (0.120 g, 0.209 mmol) followed by column chromatography
`elut.ing with 5-10% acetone in diethyl eth er provided 9 /0.054
`g, l00'Yo) as a white solid: mp 237.3 - 2:38.0 °C; [aF'n + 83.2°
`(c 1, MeOHl; 1H NMR (CD:iODl ,) 1. 35 (d, 3H, J = 22 .3 Hz),
`;3.79 i.dd, lH, J = 2.1, 12.5 Hzl, 3.94 - 4.02 (m, 3H, 5.70 (d. HI,
`J = 8.1 Hz). 6.13 (d, lH, ,J = 18.9 Hz), 8.09 (d, lH); LJC NMR
`(CD3OD) a 16.9 (d, J = 25.2 Hz), 60.l , 72. 5 (d, J = 17.6 Hz),
`83.5, 90.6 (d, J = 44 .0 Hz), 102.l (d , J = 180.1 I-lz), 103.0,
`142.0, 152.4, 166.0; 19F NMR (CD3OD) o 4.07 (bs). Anal. Calcd
`(C10H13FN2O5): C, 46.16; H, 5.04: N , 10.77 Found: C, 45 .96;
`H, 4.93; N, 10.49.
`Isolation of 2'-C-methylcytidine. Compound 7c (0.1 g,
`0.176 mmol) upon deprotection and crystallization from MeOH
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`5508 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
`
`Clark et al.
`
`gave a white solid (0.032 g, 71%): mp 244.2-245.8 °C Oit. 13
`mp 239.5-242 °C, lit.18 mp 243-245 °C); [al23o + 135. 7° (c 1,
`H2O) {lit. 18 [alo +132' (c 0.5, H 2O), lit. 19 [al2°o +128' (c 1,
`H2O)}; 1H NMR (DMSO-dGJ o 0.92 (s, lH), 3.58-3.62 (m, 2Hl,
`3.70-3.77 (m, 2H), 4.98 (s, lH), 5.06 (d, lH, J = 7.3 Hz), 5.11
`(t, lH, J = 5.0 Hz), 5.67 (d, lH, J = 7. 7 Hz), 5.87 (s, lH), 7.11
`(s, lHJ, 7.17 (s, lH), 7.94 (d, lH, J = 7.2 Hz); 13C NMR (DMSO(cid:173)
`d6) b 20.0, 59.0, 71.8, 78.3, 81.8, 91.3, 93.6, 141.1, 155.5, 165.4.
`Isolation of 2' -Deoxy-2' -methylidenecytidine (DMDC).
`Compound 7b was deprotected and crystallized from H2O: mp
`190-194 °C (dee) (transition at 90-92 °C) (lit. 20 mp 89-90
`°C); [al22D -39.8° (c 1, H20); 1H NMR (DMSO-d6) o 3.50- 3.70
`(m, 3H), 4.43 (broad t, lH), 4.93 (t, lH, J = 5.2 Hz), 5.13 (s,
`lH), 5.29 (s, lH), 5.61 (d, lH, J = 6.4 Hz), 5.69 (d, lH, J = 7.6
`Hz), 6.51 (s, lH), 7.20 (s, lH), 7.23 (s, lH), 7.47 (d, lH, J =
`7.6 Hz); 13C NMR (DMSO-d6 ) b 60.5, 69.8, 84.1, 84.2, 94.6,
`110.8, 142.0, 151.1, 155.3, 165.5.
`Biological Methods. Antiviral assays with bovine viral
`diarrhea virus and the HCV replicon were performed as
`described previously. 17
`
`Acknowledgment. Dr. R. F. Schinazi is the prin(cid:173)
`cipal founder and former director and consultant for
`Pharmasset. His laboratory received no funding for his
`participation in this work.
`
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