Synthesis of cyclopentanyl carbocyclic 5-fluorocytosine ((-)-5-fluorocarbodine) using a facially selective hydrogenation approach

Article abstract of DOI:10.1021/jo302038d

An efficient synthetic route to biologically relevant (-)-5-fluorocarbodine 6 was developed. Direct coupling of N⁶-protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the ca

Full text of DOI:10.1021/jo302038d

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Note
Synthesis of Cyclopentanyl Carbocyclic 5-Fluorocytosine ((-)-5-
Fluoro-Carbodine) Using a Facially Selective Hydrogenation Approach
Jong Hyun Cho, Franck Amblard, Steven J. Coats, and Raymond Felix Schinazi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo302038d • Publication Date (Web): 10 Dec 2012
Downloaded from http://pubs.acs.org on December 11, 2012
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Synthesis of Cyclopentanyl Carbocyclic 5ꢀ
Fluorocytosine ((ꢀ)ꢀ5ꢀFluoroꢀCarbodine) Using a
Facially Selective Hydrogenation Approach
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Jong Hyun Cho,† Franck Amblard,† Steven J. Coats,‡ Raymond F. Schinazi,*,†
† Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of
Pediatrics, Emory University School of Medicine, and Veterans Affairs Medical Center, Decatur,
GA 30033, USA
‡RFS Pharma, LLC, 1860 Montreal Road, Tucker, GA 30084, USA
Corresponding Author: rschina@emory.edu
RECEIVED DATE
ABSTRACT
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An efficient synthetic route to biologically relevant (ꢀ)ꢀ5ꢀfluorocarbodine 6 was developed.
Direct coupling of N6 protected 5ꢀfluorouracil 15 with cyclopentenyl intermediate 13, followed
by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring
closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give,
exclusively, the desired 4’ꢀβ stereoisomer.
Carbocyclic nucleosides, in which the furanose ring oxygen atom has been replaced by a carbon
atom, are, in general, more chemically and enzymatically stable and some of them have
demonstrated interesting biological properties.1 Thus, naturally occurring (ꢀ)ꢀaristeromycin (1)2
and (ꢀ)ꢀneplanocin A (2)3 manifest cytotoxic and antiviral properties while the drugs abacavir
(3)4 and entecavir (4)5 are Food and Drug Administration (FDA) approved for the treatment of
human immunodeficiency virus (HIV) and hepatitis B virus (HBV), respectively (Figure 1.).
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Figure 1. Structures of Biologically Relevant Carbocyclic Nucleosides
The carbocyclic cytosine analogs (ꢀ)ꢀcarbodine (5) and (ꢀ)ꢀ5ꢀfluorocarbodine (6) was reported to
possess significant antitumor and antiviral activities. For instance, compound (6) showed
selective submicromolar IC50’s against a human T cell lymphoblast cell line and human B cell
lymphoma cell line but also potent inhibition of HCV RNA replication (ꢁCt of 6 at 10 ꢂM in a
Huhꢀ7 cell based subgenomic replicon assay).6 In addition, compounds (5) and (6) demonstrated
selective activity against Venezuela Equine Encephalitis Virus (VEEV)7 and various strains of
H5N1 influenza.8
The most recently reported syntheses of (ꢀ)ꢀcarbodine (5) and (ꢀ)ꢀ5ꢀfluorocarbodine (6) used a
linear approach (Scheme 1) from known cyclopentenyl intermediate including stepwise
construction of the heterocyclic base. The key intermediate 7 was prepared from Dꢀribose in 13
steps by a sequence that used protecting/deprotection strategies and each step required
purification by column chromatography on silica gel. After building the pyrimidine base,
intermediate 8 was either converted, after a series of protection/deprotection, to its cytosine
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counterpart then fluorinated (ref 8), or directly fluorinated and then converted to its cytosine
analog (ref 6). Overall, both approaches are lengthy (18 and 19 steps) and resulted in the
formation of (6) in an overall yield of 26 and 10%.8
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Scheme 1. Linear Approaches for the Synthesis of 5ꢀFluorocarbodine (6)
Preparation of (ꢀ)ꢀcarbodine (5) has also been achieved by either enzymatic hydrolysis9 or
selective hydrogenation of cyclopentenyl cytosine.10 However, both of these methods resulted in
poor overall yields and low selectivities. In the later approach, the authors demonstrated that the
use of diimide as a source of hydrogen was not completely diastereoselective and led to a
mixture of 5, along with isocarbodine (10) (Scheme 2). Furthermore, separation of the two
isomers appeared problematic and purification of enantiomerically pure 5 could only be achieved
using semiꢀpreparative HPLC. With this last result in mind, we hypothesized that the
introduction of a temporary sugarꢀbase bridging group on compound A (Scheme 2; X = OH)
would act as an umbrella, covering the β face of the cyclopentene and allow for selective α face
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hydrogenation. Furthermore, we envisioned a sequence to form a cyclic eight member linker
using a ringꢀclosing metathesis (RCM)11 reaction that would connect the 5’ꢀO of the sugar and 3ꢀ
N of the uridine base as depicted in Scheme 2 compound B. Facial selectivity of the subsequent
hydrogenation could also be rationalized by the calculated12 increased ring strain of the
macrocycle for beta face hydrogenation that would ultimately lead to isocarbodine (10)
formation.
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Scheme 2. Facially Selective Hydrogenation
In order to prepare 5ꢀfluorocarbodine (6), the key cyclopentenol derivative 13 was synthesized in
9 steps from D
ꢀribose, using a slightly modified known methodology (Scheme 3).13 Thus, chiral
intermediate 11 was selectively protected by treatment with TBDPSCl in presence of DMAP to
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give compound 12. Diene 12 was then cyclized via a ringꢀclosing metathesis reaction in the
presence of catalytic amount of 2nd generation Grubbs’ catalyst D, affording desired
cyclopentenol 13. Meanwhile, N3ꢀbenzoylꢀ5ꢀfluorouracil 15 was prepared in 2 steps from 5ꢀ
fluorouracil 14 (Scheme 4) by benzoylation followed by selective deprotection using K2CO3 in
dioxane.14
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Scheme 3. Preparation of Protected Sugar 13
Coupling of chiral intermediate 13 with 3ꢀNꢀBzꢀ5ꢀFꢀuracil 15 under Mitsunobu conditions
provided the desired Nꢀalkylated derivative as a mixture with its Oꢀalkylated isomer and DIAD
byproducts (Scheme 4). The partially column purified mixture was directly deprotected by
treatmentwith 7 N NH3 in MeOH, followed by NH4F in MeOH, to afford pure carbocyclic 5ꢀ
fluorouridine derivative 17 along with pure Oꢀalkylated compound 16 (ratio 4:1) in 93% yield
over 3 steps. At this stage, introduction of pentenoyl moieties on both 5’ꢀOH and 3ꢀNH positions
of compound 17 had to be optimized. While use of pyridine either alone or with DMAP or Et3N
gave the desired compound in low yields (the monoalkylated compound being predominant), the
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use of Et3N in dichloromethane lead to the nearly quantitative formation of dialkylated
compound 18. With intermediate 18 in hand, a ring closing metathesis reaction to form the
desired macrocycle 19 was investigated. Evaluation of solvents, catalyst loading and reaction
time was undertaken. The best results were obtained when compound 18 was treated with 2.0
mol% of 2nd generation Grubbs’ catalyst D (Figure 2) in toluene at 80 °C for 1 h.
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As predicted, hydrogenation of unsaturated derivative 19 in the presence of Pd/C in EtOAc lead
to complete stereoselective formation of cyclopentane derivative 15 in 85% yield. The absolute
stereochemistry of the final product was determined by 1HꢀNMR and 1DꢀNOE experiments on
compound 21, which was obtained after removal of the linker using NH3 in MeOH. The NOE
experiment showed an enhancement of the 4’ proton when the 1’ proton was irradiated indicating
that they reside on the same face of the sugar moiety. Finally, carbocyclic 5ꢀFꢀuridine 21 was
converted to its cytosine counterpart following a previously reported method.15 The primary
alcohol in compound 21 was first protected using TBSCl. Compound 22 was then being reacted
with 2,4,6ꢀtriisopropylbenzenesulfonyl chloride followed by NH4OH to give intermediate 23.
Final deprotection was achieved using HCl in MeOH providing the target 5ꢀfluoroꢀcarbodine 6
in 98% isolated yield.
Scheme 4. Synthesis of 5ꢀFluorocarbodine 6
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CONCLUSION
In summary, an efficient synthetic route to 5ꢀfluoroꢀcarbodine 6 was developed utilizing a key
αꢀ
faceꢀselective hydrogenation driven by the introduction of a temporary macrocyclic tether. This
approach represents a noteworthy improvement to the existing syntheses of biologically relevant
compound 6 and allowed us to prepare 6 in 23% overall yield (compared with 2 to 10% yields
from previous approaches) with no major purification problems nor use of special synthetic
techniques. We are currently evaluating our umbrella approach’s compatibility with other
reactions to modify the olefin functionality and further explore the scope of this innovative
methodology.
EXPERIMENTAL SECTION
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General Considerations. Nuclear magnetic resonance (NMR) spectra (1H, C and F) were
recorded on a 400 MHz FTꢀNMR spectrometer at ambient temperature, with tetramethylsilane
(TMS) as an internal standard. Chemical shifts (δ) are reported in parts per million (ppm), and
signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd
(doublet of doublets), dt (doublet of triplets) or ddd (doublet of doublets of doublets). Highꢀ
resolution mass spectra (HRMS) were recorded on a linear ion trap LTQꢀFT mass spectrometer
with electrospray ionization (ESI). Thinꢀlayer chromatography (TLC) was performed on 0.25
mm silica gel. Purifications were carried out by silica gel column chromatography (60 Å, 63ꢀ200
ꢀm, or 40ꢀ75 ꢀm).
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(R)-1-((4R,5S)-5-(3-(tert-Butyldiphenylsilyloxy)prop-1-en-2-yl)-2,2-dimethyl-1,3-dioxolan-4-
yl)prop-2-en-1-ol (12). To a solution of compound 11 (4.0 g, 20.5 mmol) in 100 mL of
anhydrous CH2Cl2 was added DMAP (0.2 g, 1.4 mmol), TBDPSCl (6.3 g, 24.6 mmol) and Et3N
(3.1 g, 30.8 mmol) at 0 °C under N2 atmosphere. The reaction mixture was stirred for 12 h at
room temperature and quenched with 5.0 mL of MeOH. The solution was adsorbed on silica gel
and purified by silica gel column chromatography (hexane:EtOAc = 20:1 to 2:1 v/v) to give
compound 12 as a white foam (8.7 g, 19.1 mmol) in 93% yield. HRMSꢀESI+ calcd. for
1
C27H37O4Si (M+H+) 453.2462, found 453.2456; H NMR (400 MHz, CDCl3) δ 7.71ꢀ7.67 (m,
4H), 7.46ꢀ7.37 (m, 6H), 6.03ꢀ5.95 (m, 1H), 5.47 (dd, J = 13.2, 1.2 Hz, 2H), 5.32 (dt, J = 17.2, 1.2
Hz, 1H), 5.21 (dt, J = 10.4, 1.6 Hz, 1H), 4.65 (d, J = 5.6 Hz, 1H), 4.24 (s, 2H), 4.04ꢀ3.99 (m,
1H), 3.94 (dd, J = 8.8, 6.0 Hz, 1H), 2.63 (d, J = 3.2 Hz, 1H), 1.44 (s, 3H), 1.32 (s, 3H), 1.09 (s,
13
9H); C NMR (100 MHz, CDCl3) δ; 143.2, 138.0, 135.9, 135.8, 132.9, 132.8, 130.2, 130.1,
128.1, 128.0, 116.2, 113.7, 108.2, 80.9, 77.6, 70.3, 66.2, 27.5, 27.0, 25.4, 19.4.
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(3aS,4S,6aR)-6-((tert-Butyldiphenylsilyloxy)methyl)-2,2-dimethyl-4,6a-dihydro-3aH-
cyclopenta[d][1,3]dioxol-4-ol (13). To a solution of compound 12 (8.2 g, 18.1 mmol) in 500 mL
of anhydrous CH2Cl2 was added a solution of 2nd Grubb’s catalyst (0.1 mol%, 0.02 g, 0.02
mmol) in 100 mL of anhydrous CH2Cl2 at room temperature under argon atmosphere. After
stirring for 24 h, the reaction mixture was treated with 5.0 mL of DMSO, additionally stirred for
1 h at room temperature and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane:EtOAc = 10:1 to 1:1 v/v) to give compound 13 as a
white foam (7.40 g, 17.43 mmol) in 96% yield. HRMSꢀESI+ calcd. for C25H33O4Si (M+H+)
425.2149, found 425.2143; 1H NMR (400 MHz, CDCl3) δ 7.69ꢀ7.65 (m, 4H), 7.44ꢀ7.34 (m, 6H),
5.84 (d, J = 1.6 Hz, 1H), 4.86 (d, J = 5.6 Hz, 1H), 4.74 (t, J = 5.2 Hz, 1H), 4.56 (m, 1H), 4.38
(dd, J = 15.2, 20.0 Hz, 2H), 2.70 (d, J = 10.0 Hz, 1H), 1.35 (s, 3H), 1.33 (s, 3H), 1.07 (s, 9H);
13C NMR (100 MHz, CDCl3) δ 145.5, 135.7, 133.5, 129.9, 129.5, 127.9, 112.7, 83.0, 78.2, 73.5,
61.0, 27.8, 27.0, 26.9, 19.5.
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N3-Benzoyl-5-fluorouracil (15). To a solution of 5ꢀfluorouracil 14 (3.6 g, 27.3mmol) in 50 mL of
anhydrous CH3CN was added BzCl (9.3 g, 68.2 mmol), pyridine (10.8 g, 136.5mmol) at 0 °C
under N2 atmosphere. The solution was stirred for 24 h at room temperature and concentrated
under reduced pressure. The residue was dissolved in 200 mL of CH2Cl2 and then washed with
cold water (50 mL x 3) and concentrated under reduced pressure. The residue was reꢀdissolved in
50 mL of 1,4ꢀdioxane and treated with aqueous K2CO3 (20 mL, 0.5 M in water solution) at 0 °C.
After stirring for 30 min at room temperature, the pH of the solution was lowered to ca. 5.0 by
careful addition of glacial acetic acid. The solution was concentrated in vacuo and the residue
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was treated with a saturated solution of NaHCO3 (100 mL). After stirring for 1 h, the white solid
was filtered and washed with cold water (20 mL x 5) then dried under vacuum for 48 h. The
crude product was purified by silica gel column chromatography (hexane:EtOAc = 1:1 to
EtOAc) to give compound 15 as a white amorphous solid (5.6 g, 23.7 mmol) in 87% yield. MSꢀ
ESI+m/z 235 (M+H+); 1H NMR (400 MHz, DMSOꢀd6) δ 11.54 (br, 1H), 8.06 (d, J = 6.4 Hz, 1H),
8.01 (d, J = 8.4 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.58 (dd, J = 8.4, 7.6 Hz, 2H).
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5-Fluoro-2-(((3aS,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-4,6a-dihydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)oxy)pyrimidin-4(3H)-one (16) and
5-Fluoro-1-((3aS,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-4,6a-dihydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (17). To a solution of Ph3P (11.1 g,
42.4 mmol) in 150 mL of CH3CN was added DIAD (8.6 g, 42.4 mmol) at 0 °C under N2
atmosphere. After 30 min, to the reaction mixture was added a solution of compound 13 (7.2 g,
17.0 mmol) in 150 mL of CH3CN and NꢀBzꢀ5ꢀfluorouracil 15 (6.0 g, 25.4 mmol) at ꢀ78 °C. The
reaction mixture was stirred for 24 h at room temperature and then concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (hexane:EtOAc = 4:1 to
1:1 v/v) to give a non separable mixture of Nꢀ/Oꢀalkylated products along with DIAD
byproducts. This mixture was dissolved in 100 mL of MeOH and treated with 100 mL of 7 N
NH3 in MeOH at 0 °C. The solution was stirred for 12 h at room temperature and concentrated
under reduced pressure. The residue was then directly treated with a solution of ammonium
fluoride (1.6 g, 42.4 mmol) in 200 mL of MeOH at room temperature and the solution was
stirred at 50 °C for 6 h. After removal of the volatiles under vacuum, the residue was purified by
silica gel column chromatography (hexane:EtOAc = 1:5 to EtOAc v/v) to give 16 as an off white
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gum (0.94 g, 3.2 mmol) and 17 as an off white gum (3.8 g, 12.6 mmol) in 93% yield over 3
steps. Compound 16: MSꢀESI+m/z 299 (M+H+); HRMSꢀESI+calcd. for C13H16FN2O5 (M+H+)
299.1039, found 299.1035; 1H NMR (400 MHz, CD3OD) δ 7.77 (d, J = 3.2 Hz, 1H), 5.86 (d, J =
1.6 Hz, 1H), 5.72 (s, 1H), 5.19 (d, J = 5.6 Hz, 1H), 4.73 (d, J = 5.6 Hz, 1H), 4.24 (dd, J = 16.0,
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10.8 Hz, 2H), 1.38 (s, 3H), 1.34 (s, 3H); C NMR (100 MHz, CD3OD) δ 154.7, 149.3, 146.8,
136.0, 123.7, 113.7, 87.3, 85.0, 84.4, 59.00, 27.8, 26.2; Compound 17: MSꢀESI+m/z 299
(M+H+); HRMSꢀESI+calcd for C13H16FN2O5 (M+H+) 299.1038, found 299.1036; H NMR (400
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MHz, CD3OD) δ 7.54 (d, J = 6.4 Hz, 1H), 5.60 (m, 1H), 5.36 (s, 1H), 5.24 (d, J = 6.0 Hz, 1H),
4.62 (d, J = 5.6 Hz, 1H), 4.29 (s, 2H), 1.40 (s, 3H), 1.33 (s, 3H); 13C NMR (100 MHz, CD3OD) δ
155.0, 151.5, 143.1, 140.8, 127.8, 122.6, 113.5, 85.6, 84.8, 68.9, 60.0, 27.7, 26.1; 19F NMR (376
MHz, CD3OD) δ ꢀ169.69 (d, J = 6.0 Hz).
((3aR,6R,6aS)-6-(3-But-3-enoyl-5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-
dimethyl-6,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)methyl but-3-enoate (18). To a
solution of compound 17 (1.4 g, 4.7mmol) in 40 mL of anhydrous CH2Cl2 was added Et3N (2.4
g, 23.5 mmol) and 4ꢀpentenoyl chloride (1.7 g, 14.1 mmol) at 0 °C under argon atmosphere.
After stirring for 4 h at room temperature, the solution was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (hexane:EtOAc = 4:1 to 1:1 v/v)
to give a compound 18 as a white foam (2.1 g, 4.5 mmol) in 97% yield. MSꢀESI+m/z 463
(M+H+); HRMSꢀESI+calcd for C23H27FN2O7Na (M+Na+) 485.1705, found 485.1696; H NMR
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(400 MHz, CDCl3) δ 7.09 (d, J = 5.6 Hz, 1H), 5.90ꢀ5.78 (m, 2H), 5.57 (s, 1H), 5.39 (s, 1H), 5.21
(d, J = 5.6 Hz, 1H), 5.15ꢀ5.01 (m, 4H), 4.82 (q, J = 15.2 Hz, 2H), 4.59 (d, J = 5.6 Hz, 1H), 2.97
(t, J = 7.2 Hz, 2H), 2.51 (m, 4H), 2.43 (m, 2H), 1.43 (s, 3H), 1.35 (s, 3H); 13C NMR (100 MHz,
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CDCl3) δ 173.9, 172.8, 158.7, 149.3, 148.0, 136.6, 135.6, 124.9, 122.9, 116.5, 116.1, 113.4, 94.6,
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84.1, 83.5, 68.1, 60.9, 39.8, 33.4, 28.9, 27.5, 27.4, 25.9; F NMR (376 MHz, CDCl3) δ ꢀ165.50
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(d, J = 4.5 Hz).
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Compound 19. To a solution of compound 18 (1.0 g, 2.16 mmol) in 500 mL of anhydrous
toluene was added 2nd Generation Grubbs’ catalyst D (2.0 mol%, 0.037 g, 0.043 mmol) at room
temperature under an argon atmosphere. After stirring for 1 h at 80 °C, the reaction mixture was
treated with 2.0 mL of DMSO at room temperature and additionally stirred for 1 h. The solution
was concentrated under reduced pressure and then purified by silica gel column chromatography
(hexane:EtOAc = 2:1 v/v) to give compound 19 as a white foam (0.85 g, 2.0 mmol) in 90%
yield. MSꢀESI+m/z 435 (M+H+); HRMSꢀESI+calcd for C21H24FN2O7 (M+H+) 435.1562, found
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435.1560; H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 5.2 Hz, 1H), 5.62 (s, 1H), 5.56ꢀ5.40 (m,
3H), 5.25 (d, J = 11.6 Hz, 1H), 4.90 (d, J = 5.6 Hz, 1H), 4.42 (s, 1H), 4.25 (d, J = 11.2 Hz, 1H),
3.07 (ddd, J = 18.0, 8.0, 2.4 Hz, 1H), 2.86 (ddd, J = 18.0, 10.4, 2.4 Hz, 1H), 2.60 (m, 1H), 2.50ꢀ
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2.30 (m, 3H), 2.28ꢀ2.15 (m, 2H), 1.40 (s, 3H), 1.36 (s, 3H); C NMR (100 MHz, CDCl3) δ
174.5, 172.7, 155.9, 146.9, 141.1, 138.7, 129.8, 129.1, 127.7, 125.5, 112.3, 84.5, 82.7, 74.9, 58.6,
39.4, 32.8, 27.4, 25.8; 19F NMR (376 MHz, CDCl3) δ ꢀ165.63 (d, J = 6.0 Hz), ꢀ166.20 (d, J = 6.0
Hz).
Compound 20. To a solution of compound 19 (2.5 g, 5.8 mmol) in 100 mL of EtOAc was added
Pd/C catalyst (10 wt %, 0.25 g). The solution was stirred for 12 h under H2 atmosphere (1 atm)
and then treated with Celite. After stirring for 30 min, the suspension was carefully filtered, the
filtrate concentrated under vacuum, and then purified by silica gel column chromatography
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(hexane:EtOAc = 1:1 v/v) to give compound 20 as a white foam (2.1 g, 4.9 mmol) in 85% yield.
MSꢀESI+m/z 439 (M+H+); HRMSꢀESIꢀcalcd for C21H27FN2O7Cl (M+Clꢀ) 473.1496, found
473.1499; 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 5.6 Hz, 1H), 5.00 (dd, J = 6.4, 2.4 Hz, 1H),
4.88 (t, J = 5.6 Hz, 1H), 4.70 (d, J = 12.0, 1.2 Hz, 1H), 3.99 (dd, J = 12.0, 1.2 Hz, 1H), 3.91 (m,
1H) 2.95 (ddd, J = 16.0, 8.4, 3.2 Hz, 1H), 2.73 (ddd, J = 16.0, 9.6, 3.2 Hz, 1H), 2.55 (ddd, J =
16.0, 8.4, 5.2 Hz, 1H), 2.33ꢀ2.17 (m, 4H), 1.90 (m, 1H), 1.79 (s, 1H), 1.66ꢀ1.40 (m, 6H), 1.39ꢀ
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1.25 (m, 6H); C NMR (100 MHz, CDCl3) δ 174.4, 174.1, 147.6, 141.1, 138.7, 130.1, 129.8,
113.0, 94.6, 83.4, 80.3, 69.7, 61.0, 45.7, 40.1, 33.6, 31.2, 28.1, 27.1, 25.6, 24.4; 19F NMR (376
MHz, CDCl3) δ ꢀ166.59 (d, J = 4.5 Hz),
5-Fluoro-1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (21). A solution of compound 20
(2.0 g, 4.58 mmol) in 50 mL of MeOH was treated with 20 mL of 7 N NH3 in MeOH at 0 °C in a
sealed tube. After stirring for 12 h at 80 °C, the solution was concentrated under reduced
pressure and the residue was purified on a silica gel pad (CH2Cl2:MeOH = 30:1 to 10:1 v/v) to
give compound 21 as a beige gum (1.3 g, 4.3 mmol) in 94% yield. MSꢀESI+m/z 301 (M+H+);
1
HRMSꢀESI+calcd for C13H18FN2O5 (M+H+) 301.1194, found 301.1193; H NMR (400 MHz,
CDCl3) δ 10.01 (br, 1H), 7.48 (d, J = 5.6 Hz, 1H), 4.77 (dd, J = 7.2, 5.6 Hz, 1H), 4.61 (dd, J =
7.2, 4.8 Hz, 1H), 4.57 (m, 1H), 3.83 (dd, J = 10.4, 4.8 Hz, 1H), 3.74 (dd, J = 10.8, 4.8 Hz, 1H),
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2.80 (br, 1H) 2.37ꢀ2.27 (m, 2H), 2.12ꢀ2.03 (m, 1H), 1.54 (s, 3H), 1.31 (s, 3H); C NMR (100
MHz, CDCl3) δ 157.5, 157.2, 149.8, 141.9, 139.6, 127.9, 127.6, 113.2, 83.0, 81.4, 64.2, 63.3,
45.6, 32.0, 27.7, 25.4; 19F NMR (376 MHz, CDCl3) δ ꢀ169.67 (d, J = 6.0 Hz).
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1-((3aS,4R,6R,6aR)-6-((tert-Butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (22). To a solution of
compound 21 (1.3 g, 4.5 mmol) in 30 mL of anhydrous CH2Cl2 was added imidazole (0.74 g,
10.9mmol) and TBDPSCl (1.7 g, 5.4 mmol) at 0 °C under N2 atmosphere. The reaction mixture
was stirred for 12 h at room temperature and treated with 5.0 mL of MeOH. After stirring for 1 h
at room temperature, the solution was concentrated under reduced pressure and purified by silica
gel column chromatography (hexane:EtOAc = 2:1 to 1:4 v/v) to give compound 22 as an off
white foam (2.3 g, 4.3mmol) in 98% yield. MSꢀESI+m/z 539 (M+H+);HRMSꢀESI+calcd for
C29H36FN2O5Si (M+H+) 539.2372, found 539.2368; 1H NMR (400 MHz, CDCl3) δ 9.78 (br, 1H),
7.66ꢀ7.62 (m, 4H), 7.40ꢀ7.36 (m, 6H), 7.23 (d, J = 5.6 Hz, 1H), 4.71 (m, 1H), 4.63 (dd, J = 6.8,
6.0 Hz, 1H), 4.51 (dd, J = 7.2, 6.8 Hz, 1H), 3.79 (d, J = 5.2 Hz, 2H), 2.33 (m, 1H), 2.25 (m, 1H),
2.00 (q, J = 12.0 Hz, 1H), 1.53 (s, 3H), 1.29 (s, 3H), 1.09 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
157.2, 157.0, 149.7, 142.1, 139.7, 135.83, 135.78, 133.5, 133.4, 130.1, 128.0, 126.6, 126.3,
113.9, 82.8, 80.7, 64.3, 63.8, 45.2, 32.4, 27.8, 27.2, 25.5, 19.6; 19F NMR (376 MHz, CDCl3) δ ꢀ
165.85 (d, J = 4.5 Hz).
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4-Amino-1-((3aS,4R,6R,6aR)-6-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyltetrahydro-
3aH-cyclopenta[d][1,3]dioxol-4-yl)-5-fluoropyrimidin-2(1H)-one (23). To a solution of
compound 22 (2.2 g, 4.1 mmol) and DMAP (2.0 g, 16.4 mmol) in 50 mL of anhydrous CH3CN
was added 2,4,6ꢀtrisiopropyl benzenesulfonyl chloride (3.1 g, 10.3mmol) and Et3N (1.7 g, 16.4
mmol) at room temperature under N2 atmosphere. The reaction mixture was stirred for 48 h at
room temperature and treated with 25 mL of 30% NH4OH solution. After stirring for 4 h at room
temperature, the solution was poured into 150 mL of a cold waterꢀCHCl3 solution (1:2 v/v) and
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then extracted with CHCl3 (30 mL x 3). The combined organic layers were washed with a
saturated NH4Cl aqueous solution (100 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2 to
CH2Cl2:MeOH = 10:1 v/v) to give compound 23 as a gum (2.0 g, 3.7mmol) in 89% yield. MSꢀ
ESI+m/z 538 (M+H+); HRMSꢀESI+calcd for C29H37FN3O4Si (M+H+) 538.2532, found 538.2525;
1H NMR (400 MHz, CDCl3) δ 8.83, (br, 1H), 7.66ꢀ7.63 (m, 4H), 7.45ꢀ7.36 (m, 6H), 7.24 (d, J =
5.6 Hz, 1H), 5.60 (br, 1H), 4.80 (dd, J = 7.2, 5.2 Hz, 1H), 4.52ꢀ4.44 (m, 2H), 3.82 (dd, J = 10.0,
4.4 Hz, 1H), 3.74 (dd, J = 10.0, 6.0 Hz, 1H), 2.33ꢀ2.25 (m, 2H), 2.16 (m, 1H), 1.50 (s, 3H), 1.26
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(s, 3H), 1.07 (s, 9H); C NMR (100 MHz, CDCl3) δ 158.1, 154.6, 137.8, 135.8, 135.7, 135.4,
133.7, 133.6, 129.9, 128.4, 128.1, 127.9, 113.3, 83.0, 81.1, 66.1, 64.8, 46.2, 32.8, 27.8, 27.1,
25.5, 19.5; 19F NMR (376 MHz, CDCl3) δ ꢀ169.48 (d, J = 7.5 Hz).
4-Amino-1-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-5-fluoropyrimidin-
2(1H)-one (6). A solution of compound 23 (2.5 g, 4.7mmol) in 100 mL of MeOH was treated
with 50 mL of HCl in MeOH at 0 °C. After stirring for 3 h at room temperature, the acidic
solution was concentrated under reduced pressure. The residue was dissolved in water (50 mL)
and washed with EtOAc (20 mL x 5). The aqueous layer was concentrated under reduced
pressure. The residue was dissolved in EtOH (10 mL) and the solution was concentrated under
reduced pressure. After this process was repeated 5 times, the final residue was dried under
vacuum for 72 h at room temperature to give pure compound 6 as a white amorphous solid (1.4
g, 4.6 mmol) as a HCl salt in 98% yield. MSꢀESI+m/z 260 (M+H+); HRMSꢀESI+calcd for
C10H15FN3O4 (M+H+) 260.1041, found 260.1039; 1H NMR (400 MHz, CD3OD) δ 8.35 (d, J =
7.2 Hz, 1H), 4.70 (q, J = 9.2 Hz, 1H), 4.12 (dd, J = 5.6, 9.2 Hz, 1H), 3.85 (dd, J = 5.6, 3.2 Hz,
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1H), 3.53 (d, J = 5.2 Hz, 2H), 2.19 (m, 1H), 2.05 (m, 1H), 1.46 (m, 1H); C NMR (100 MHz,
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CD3OD) δ 154.65, 154.42, 148.52, 139.97, 137.78, 133.74, 133.46, 75.44, 73.49, 64.83, 64.53,
46.50, 28.67; 1H NMR (400 MHz, DMSOꢀd6) δ 9.75 (br, 1H), 9.14 (br, 1H), 8.53 (d, J = 7.2 Hz,
1H), 5.32 (br, 3H), 4.65 (q, J = 9.2 Hz, 1H), 4.01 (dd, J = 9.2, 5.6 Hz, 1H), 3.72 (dd, J = 4.8, 2.4
Hz, 1H), 3.42 (dd, J = 10.4, 6.4 Hz, 1H), 3.35 (dd, J = 10.4, 5.6 Hz, 1H), 2.04 (m, 1H), 1.93 (m,
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1H), 1.28 (m, 1H); C NMR (100 MHz, DMSOꢀd6) δ 153.1, 152.9, 147.8, 136.2, 133.9, 131.8,
131.5, 73.4, 71.2, 62.9, 61.8, 44.81, 27.8; 19F NMR (376 MHz, CD3OD) δ ꢀ170.99 (d, J = 7.52
Hz).
ACKNOWLEDGMENTS
This work was supported in part by NIH grant 5P30ꢀAIꢀ50409 (CFAR) and by the Department
of Veterans Affairs. Dr. Schinazi is the founder and a major shareholder of RFS Pharma, LLC.
Emory received no funding from RFS Pharma, LLC to perform this work and vice versa.
Supporting Information
Copies of NMR spectra. This material is available free of charge via the Internet at
http://pubs.acs.org.
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