Synthesis and antiviral evaluation of fluorinated acyclo-nucleosides and their phosphoramidates

Article abstract of DOI:10.1080/15257770.2016.1218023

A novel series of tetrafluoro and hexafluoro acyclic nucleosides and their phosphoramidates were successfully prepared from commercially available 2,2,3,3-tetrafluoro-1,4-butanediol and 2,2,3,3,4,4-hexafluoro-1,5-pentanediol in four to six steps. Their ab

Full text of DOI:10.1080/15257770.2016.1218023

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and antiviral evaluation of fluorinated
acyclo-nucleosides and their phosphoramidates
Sawsan Mahmoud, Hao Li, Tamara R. McBrayer, Leda Bassit, Sherif F.
Hammad, Steven J. Coats, Franck Amblard & Raymond F. Schinazi
To cite this article: Sawsan Mahmoud, Hao Li, Tamara R. McBrayer, Leda Bassit, Sherif
F. Hammad, Steven J. Coats, Franck Amblard & Raymond F. Schinazi (2016): Synthesis
and antiviral evaluation of fluorinated acyclo-nucleosides and their phosphoramidates,
Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2016.1218023
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1218023
Published online: 19 Oct 2016.
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Date: 20 October 2016, At: 04:40

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Synthesis and antiviral evaluation of fluorinated
acyclo-nucleosides and their phosphoramidates
Sawsan Mahmouda,b, Hao Lia, Tamara R. McBrayerc, Leda Bassita, Sherif F. Hammadb,
Steven J. Coatsc, Franck Amblarda, and Raymond F. Schinazia
aCenter for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory
University School of Medicine, Atlanta, Georgia, USA; bDepartment of Pharmaceutical Chemistry, Faculty
of Pharmacy, Helwan University, Helwan, Egypt; cCocrystalPharma, Inc., Tucker, Georgia, USA
ABSTRACT
ARTICLE HISTORY
Received  April 
Accepted  July 
A novel series of tetrafluoro and hexafluoro acyclic nucleo-
sides and their phosphoramidates were successfully prepared
fromcommerciallyavailable2,2,3,3-tetrafluoro-1,4-butanedioland
2,2,3,3,4,4-hexafluoro-1,5-pentanediol in four to six steps. Their
ability to block HIV, HCV, HSV-1, and HBV replication along with
their cytotoxicity toward HepG2, human lymphocyte, CEM, and
Vero cells was assessed.
KEYWORDS
Fluorine; acyclonucleoside;
phosphoramidates; antiviral
agent
Introduction
Since the discovery of acyclovir in 1977,[1] acyclic nucleosides have taken a pre-
dominant place in our arsenal against viral diseases, especially against herpes virus
infections. Thus, multiple acyclic nucleosides such as acyclovir 1 (in the form of pro-
drugvalacyclovir, 2), famciclovir 3, ganciclovir 4, and penciclovir 5 were approved
for the treatment of herpes simplex virus (HSV), varicella zoster virus (VZV), and
cytomegalovirus (CMV) while acyclic phosphonates such as tenofovir 6a (in the
form of prodrug TDF, 6b) and adefovir 7 are commonly used for the treatment of
human immunodeficiency virus (HIV) and hepatitis B virus (HBV) (Figure 1).[2]
More recently, tenofoviralafenamidefumarate (TAF, GS-7340, 6c), a new prodrug of
tenofovir, with improved toxicity profile has been developed for the treatment of
HIV and HBV.
Based on its unique stereoelectronic properties, fluorine is often used in medic-
inal chemistry to mimic the size of a proton or the polarity of a hydroxyl group.
In addition, because fluorine is the most electronegative atom in the periodic table
(4.0 on the Pauling scale), its incorporation into a molecule results in considerable
electronic changes in the molecule dipole moment and the acidity or basicity of
other groups nearby. Thus, fluorinated nucleosides have attracted a great deal of
CONTACT Raymond F. Schinazi
rschina@emory.edu
Center for AIDS Research, Laboratory of Biochemical
Pharmacology, Department of Pediatrics, Emory University School of Medicine,  Haygood Drive, Room E, Atlanta,
GA , USA.
©  Taylor & Francis Group, LLC

2
S. MAHMOUD ET AL.
Figure . Structures of clinically relevant acyclic nucleosides/nucleotides.
attention,[3] which culminated with the approval of compounds such as capecitabine
8 and gemcitabine 9 (cancer), emtricitabine (FTC 10; HIV), and more recently
sofosbuvir 11 (hepatitis C virus, HCV) (Figure 2). Herein we report the synthesis
and the antiviral evaluation of new highly fluorinated acyclonucleosides and phos-
phoramidates.
Result and discussion
Commercially available 2,2,3,3-tetrafluoro-1,4-butanediol 12a and 2,2,3,3,4,4-
hexafluoro-1,5-pentanediol 12b was first protected using tert-butyldipheny-
lchlorosilane (TBDPSCl) in presence of imidazole to give compound 13a–b in 39
and 40% yield then treated with Tf2O in pyridine to give compound 14a–b in 90 and
98% yield. Reaction of triflate intermediates 14a–b with either N-3-benzoyl-uracil,
N-3-benzoyl-thymine, N-4-benzoyl-cytosine, adenine, or 2-amino-6-chloropurine
in presence of Cs2CO3 led to the formation of compounds 15a–b through 19a–b
respectively in yields ranging from 35% to 92% (Scheme 1).
Uracil, thymine, and cytosine acyclonucleosides 23–25 were obtained in excellent
yields after deprotection of compounds 15–17 using first ammonia in MeOH and
then tBuNH4+F− in THF (Scheme 2). Adenosine analogs 26a–b were obtained by
treatment of compounds 18a–b with tBuNH4+F− in THF (Scheme 3). On the other
hand, guanine analogs 27a–b were obtained by heating under reflux compounds
19a–b in 80% acetic acid (Scheme 4).
It is well established that certain nucleoside analogs are unable to be intracel-
lularly metabolized to their corresponding nucleoside triphosphates. Therefore, in
order to overcome the often rate-limiting first phosphorylation step and poten-
tially improve the antiviral activity of our nucleosides analogs, we prepared their
corresponding monophosphate McGuigan type prodrugs.[4] Ethyl and isopropyl

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . FDA approved fluorinated nucleosides 9–11 and targeted fluorinated acyclonucleosides.
Scheme . Synthesis of compounds 15–19. Reagents and conditions: (a) TBDPSCl, imidazole, rt,
CHCl, –%; (b) TfO, rt, pyridine, –%; (c) N-Bz-uracil, N-Bz-thymine, N-Bz-cytosine,
-amino--chloropurine or adenine, CsCO, rt, – hours, DMF, –%.

4
S. MAHMOUD ET AL.
Scheme . Synthesis of compounds 23–25. Reagents and conditions: (a) NH, CHOH, rt,  hours,
–%; (b) tBuNH+F−, THF, rt, – hours, –%.
Scheme . Synthesis of compounds 26a–b. Reagents and conditions: tBuNH+F−, THF, rt,  hours,
–%.
phosphoramidateprodrugs 29–33 and 35–39 were prepared according to the chem-
istry described in Scheme 5. Thus, uracil, thymine, cytosine, and guanine pro-
drugs 29, 35, 30, 36, 31, 37, 33, and 39 were prepared from 23–25 and 27 by reac-
tion with either ethyl (chloro(phenoxy)phosphoryl)-L-alaninate 28[5] or isopropyl
(chloro(phenoxy)phosphoryl)-L-alaninate 34[6] in presence of tBuMgCl. On the
other hand, adenine analogs 32 and 38 were prepared using N-methylimidazole.
Notably, in order to increase the solubility of starting material 27, and thus facilitate
the formation of the prodrug, a mixture of THF/DMF (4:1) was used as solvent in
the synthesis of prodrugs 33 and 39.
Biology
Anti-HIV, HBV, HSV-1[7], and HCV (subgenomic HCV replicon system) potency
of compounds 23–27 and phosphoramidateprodrugs 29–33 and 35–39 were
Scheme . Synthesis of compounds 27a–b. Reagents and conditions: % acetic acid, °C,  hours,
–%.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme . Synthesis of prodrugs29–33 and 35–39. Reagents and conditions: (a) For 29–31, 35–37:
phosphoramidate reagent 28 or 34, tBuMgCl, THF, rt, – hours, –%; (b) For 32, 38: phospho-
ramidate reagent 28 or 34, NMI, THF, rt, – hours, %; (c) for 33, 39: phosphoramidate reagent 28
or 34, tBuMgCl, THF/DMF :, rt, – hours, –%.
evaluated in primary human lymphocytes, HepG2, Vero, and Huh-7 cells, respec-
tively. Zidovudine (AZT), lamivudine (3TC), acyclovir, and 2ꢀ-C-methyl-cytidine
were used as positive controls. In addition, cytotoxicity was determined in human
peripheral blood mononuclear (PBM) cells, human lymphoblastoid CEM, African
Green monkey Vero cells, and HepG2 cells.[8] Except for compounds 30 and 32,
which were found to exhibit moderate anti-HIV activity (EC50 = 44 and 31 μM
respectively), none of the other nucleoside analogs or phosphoramidates displayed
significant antiviral activity nor cytotoxicity when evaluated up to 100 μM.
Conclusion
A novel series of (2,2,3,3-tetrafluoro-4-hydroxybutyl) and (2,2,3,3,4,4-hexafluoro-
5-hydroxypentyl) acyclonucleosides and their phosphoramidates were prepared in
four to six steps from commercially available diols 12a and 12b. Unfortunately, none
showed significant activity versus HIV, HBV, HSV-1, or HCV.
Experimental
General
Nuclear magnetic resonance (NMR) spectra (1H,13C, 19F, and31P) were recorded on
a Bruker AscendTM 400 MHz Fourier transform spectrometer at rt, with tetram-
ethylsilane (TMS) as an internal standard. Chemical shifts (δ) are reported in parts

6
S. MAHMOUD ET AL.
per million (ppm), and signals are quoted as s (singlet), d (doublet), t (triplet),
q (quartet), p (pentet), m (multiplet), dd (doublet of doublets), ddd (doublet of dou-
blets of doublets), dt (doublet of triplets), or td (triplet of doublets). The phospho-
ramidates are an approximate 50:50 mixtures of diastereomers (RP/SP) and the13C
NMR data are reported as observed, that is, some carbon signals overlap with solvent
signals. High-resolution mass spectra (HRMS) were recorded on a ThermoFisher
Q exactive or Thermo (LTQ-FTMS) Plus high-resolution mass spectrometer with
electrospray ionization (ESI). Thin-layer chromatography (TLC) was performed on
0.25 mm silica gel. Purifications were carried out on silica gel column chromatog-
˚
raphy (60 A, 63–200 μm, or 40–75 μm).
4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutan-1-ol (13a)
To a solution of compound 12a (2.30 mmol, 372 mg) in dry CH2Cl2 (5 mL) at
0°C were added imidazole (4.70 mmol, 313 mg, 2 eq.) and TBDPSCl (2.30 mmol,
0.6 mL, 1 eq.). The mixture was stirred overnight and evaporated under reduced
pressure. The residue was purified by silica gel column chromatography eluting with
hexane:ethyl acetate (80:20) to afford compound 13a (0.92 mmol, 370 mg, 40%) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.90 – 7.81 (m, 4H), 7.55 (q, J = 7.3, 6.2 Hz,
6H), 4.19 (t, J = 14.4 Hz, 4H), 3.45 (s, 1H), 1.25 (s, 9H). 13C NMR (101 MHz, CDCl3)
δ 135.69, 131.95, 130.37, 128.13, 118.86 (td, J = 31.0, 9.3 Hz), 116.35 (td, J = 31.1,
9.7 Hz), 113.84 (td, J = 31.3, 10.2 Hz), 61.78 (t, J = 28.6 Hz), 60.67 (t, J = 26.0 Hz),
26.69, 19.36. 19F NMR (377 MHz, CDCl3) δ −124.23 (t, J = 13.3 Hz), −125.58
(t, J = 14.5 Hz). MS (Thermo LTQ-FTMS): m/z [M+Na]+ calcd. for
C20H24F4NaO2Si: 423.1373, found: 423.1377.
5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentan-1-ol (13b)
Compound 13b was prepared from 12b using the same procedure as for compound
13a; yield 39%. 1H NMR (400 MHz, CDCl3) δ 7.94 – 7.86 (m, 4H), 7.64 – 7.53 (m,
6H), 4.31 – 4.17 (m, 4H), 3.66 (d, J = 6.6 Hz, 1H), 1.30 (s, 9H). 13C NMR (101 MHz,
CDCl3) δ 135.59, 132.06, 130.15, 127.95, 118.26 (t, J = 30.3 Hz), 115.73 (t, J =
30.3 Hz), 114.38 (t, J = 32.6 Hz), 113.19 (t, J = 30.1 Hz), 111.77 (t, J = 32.7 Hz),
109.17 (t, J = 32.6 Hz), 61.78 (t, J = 26.8 Hz), 60.70 (t, J = 25.1 Hz), 26.50, 19.32. 19F
NMR (377 MHz, CDCl3) δ −122.34 (p, J = 12.0 Hz)p), −124.07 (p, J = 14.3 Hz),
−126.81. MS (Thermo LTQ-FTMS): m/z [M+Na]+ calcd. for C21H24F6NaO2Si:
473.1342, found: 473.1341.
4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl trifluoromethanes-
ulfonate(14a)
To a solution of compound 13a (0.60 mmol, 240 mg) in dry CH2Cl2 (1 mL) at −20°C
were added pyridine (1.70 mmol, 0.1 mL, 2.8 eq.) and Tf2O (0.7 mmol, 0.1 mL,
1.15 eq.). The mixture was stirred for 2 hours at −20°C and quenched with 5%
HCl (3 mL). The organic layer was separated and washed with 5% HCl (5 mL),
H2O (5 mL) and brine (5 mL), dried over sodium sulfate and concentrated under
reduced pressure to afford compound 14a (0.54 mmol, 287 mg, 90%) as a yellow

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
oil. 1H NMR (400 MHz, CDCl3) δ 8.00 – 7.88 (m, 4H), 7.68 – 7.55 (m, 6H), 5.20
(t, J = 13.8 Hz, 2H), 4.29 – 4.19 (m, 2H), 1.33 (s, 9H). 13C NMR (101 MHz, CDCl3) δ
135.66, 131.48, 130.55, 128.26, 123.51, 120.34, 118.43 (t, J = 29.8 Hz), 117.16, 116.55
– 115.41 (m), 114.31 – 113.23 (m), 70.13 (t, J = 24.4 Hz), 61.66 (t, J = 30.0 Hz), 26.56,
19.24. 19F NMR (377 MHz, CDCl3) δ −75.75, −123.73 (t, J = 12.3 Hz), −123.91 (t,
J = 14.0 Hz). MS (Thermo LTQ-FTMS): m/z [M+Cl]− calcd. for C21H23ClF7O4SSi:
567.0668, found: 567.0679.
5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyltrifluoromethane
sulfonate(14b)
Compound 14b was prepared from 13b using the same procedure as for compound
1
14a; yield 98%. H NMR (400 MHz, CDCl3) δ 7.86 – 7.76 (m, 4H), 7.59 – 7.49
(m, 6H), 4.91 (t, J = 12.9 Hz, 2H), 4.17 (t, J = 13.4 Hz, 2H), 1.20 (s, 9H). 13C NMR
(101 MHz, CDCl3) δ 135.59, 131.83, 130.29, 128.03, 123.29, 120.11, 116.94, 115.66 (t,
J = 29.3 Hz), 114.04 – 112.66 (m), 68.93 (t, J = 26.1 Hz), 61.71 (t, J = 27.4 Hz), 26.44,
19.29. 19F NMR (377 MHz, CDCl3) δ −75.58, −121.74 (p, J = 11.8 Hz), −121.99
(p, J = 11.6, 11.0 Hz), −126.33. MS (Thermo LTQ-FTMS): m/z [M+Cl]− calcd. for
C22H23ClF9O4SSi: 617.0636, found: 617.0643.
3-Benzoyl-1-(4-((tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)
pyrimidine-2,4(1H,3H)-dione (15a)
To a solution of compound 14a (0.50 mmol, 266 mg) in anhydrous DMF (2mL) were
added N-3-benzoyl-uracil (0.60 mmol, 130 mg, 1.2 eq.) and Cs2CO3 (0.60 mmol,
195 mg, 1.2 eq.) at room temperature. The mixture was stirred for 5 hours at
room temperature and quenched by addition water (4 mL). The aqueous layer was
extracted with ethyl acetate (3 × 5 mL) and the combined organic layers were
washed with brine (5 mL) and dried over Na2SO4. After concentrated under vac-
uum the residue was purified by silica gel column chromatography eluting with
dichloromethane/methanol (95:5) to afford compound 15a (0.30 mmol, 180 mg,
60%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.00 – 7.39 (m, 15H), 7.31
(d, J = 8.1 Hz, 1H), 5.90 (d, J = 8.1 Hz, 1H), 4.54 (t, J = 15.3 Hz, 2H), 4.11 –
4.01 (m, 2H), 1.10 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 168.14, 161.89, 149.70,
143.97, 135.54, 135.26, 131.66, 131.23, 130.48, 130.29, 129.25, 128.04, 103.08, 61.33,
46.61, 26.62, 19.27.19F NMR (377 MHz,CDCl3) δ −119.96, −123.58 (t, J = 13.3 Hz).
HRMS (ESI): m/z [M+H]+ calcd. for C31H30F4N2O4Si: 599.1989, found: 599.1986.
3-Benzoyl-1-(5-((tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)
pyrimidine-2,4(1H,3H)-dione(15b)
Compound 15b was prepared from 14b using the same procedure as for compound
1
15a; yield 63%. H NMR (400 MHz, CDCl3) δ 8.05 – 7.40 (m, 15H), 7.31 (d, J
= 8.1 Hz, 1H), 5.88 (d, J = 8.1 Hz, 1H), 4.49 (t, J = 15.4 Hz, 2H), 4.11 (t, J =
13.6 Hz, 2H), 1.13 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 168.13, 161.83, 149.70,
143.93, 135.56, 135.41, 131.89, 131.15, 130.43, 130.26, 129.34, 128.02, 103.22, 61.80
(t, J = 27.4 Hz), 46.48 (t, J = 22.0 Hz), 26.52, 19.34. 19F NMR (377 MHz, CDCl3) δ

8
S. MAHMOUD ET AL.
−118.88, −121.68 (p, J = 12.1 Hz), −126.52.HRMS (ESI): m/z [M+H]+ calcd. for
C32H30F6N2O4Si: 649.1957, found: 649.1948.
3-Benzoyl-1-(4-((tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)-5-
methylpyrimidine2,4(1H,3H)-dione (16a)
Compound 16a was prepared from 14a using the same procedure as for compound
15a; yield 51%. 1H NMR (400 MHz, CDCl3) δ 8.00 – 7.36 (m, 15H), 7.23 – 7.08 (m,
1H), 4.51 (t, J = 15.5 Hz, 2H), 4.06 (t, J = 13.1 Hz, 2H), 2.01 (d, J = 1.3 Hz, 3H),
1.11 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 168.49, 162.81, 149.88, 139.88, 135.56,
135.16, 131.73, 131.42, 130.45, 130.29, 129.22, 128.05, 111.61, 61.35 (t, J = 28.6 Hz),
46.42 (t, J = 22.1 Hz), 26.63, 19.29, 12.43. 19F NMR (377 MHz, CDCl3) δ −119.87,
−123.63 (t, J = 13.2 Hz). HRMS (ESI): m/z [M+H]+ calcd. for C32H32F4N2O4Si:
613.2146, found: 613.2143.
3-Benzoyl-1-(5-((tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (16b)
Compound 16b was prepared from 14b using the same procedure as for compound
15a; yield 42%. 1H NMR (400 MHz,CDCl3) δ 7.97 – 7.38 (m, 15H), 7.13 (s, 1H),
4.45 (t, J = 15.3 Hz, 2H), 4.06 (t, J = 13.6 Hz, 2H), 2.01 (d, J = 1.2 Hz, 3H), 1.08 (s,
9H). 13C NMR (101 MHz, CDCl3) δ 168.14, 162.61, 149.78, 139.24, 135.52, 135.14,
131.87, 131.32, 130.43, 130.18, 129.20, 127.95, 112.09, 26.47, 19.29, 12.47.19F NMR
(377 MHz, CDCl3) δ −118.85, −121.74 (p, J = 12.0 Hz), −126.64. HRMS (ESI): m/z
[M+H]+ calcd. for C33H32F6N2O4Si: 663.2114, found: 663.2104.
N-(1-(4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (17a)
Compound 17a was prepared from 14a using the same procedure as for compound
15a; yield 35%. 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 7.95 (d, J = 7.6 Hz, 2H),
7.77 – 7.25 (m, 15H), 4.73 (t, J = 15.8 Hz, 2H), 4.08 (t, J = 13.1 Hz, 2H), 1.10 (s,
9H). 13C NMR (101 MHz, CDCl3) δ 162.83, 149.10, 135.56, 133.32, 131.76, 130.23,
129.07, 128.02, 127.65, 61.36 (t, J = 28.8 Hz), 47.65 (t, J = 21.8 Hz), 26.60, 19.26.
19F NMR (377 MHz, CDCl3) δ −119.92 (t, J = 16.1 Hz), −123.40 (t, J = 13.0 Hz).
HRMS (ESI): m/z [M+H]+ calcd. for C31H31F4N3O3Si: 598,2149, found: 598.2143.
N-(1-(5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide (17b)
Compound 17b was prepared from 14b using the same procedure as for compound
15a; yield 37%. 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.11 – 7.38 (m, 17H),
4.68 (t, J = 15.8 Hz, 2H), 4.09 (t, J = 13.7 Hz, 2H), 1.10 (s, 9H). 13C NMR (101 MHz,
CDCl3) δ 163.15, 148.96, 135.55, 133.29, 131.94, 130.18, 129.00, 127.97, 127.77,
62.23 – 61.34 (m), 47.85 – 47.28 (m), 26.51, 19.31. 19F NMR (377 MHz, CDCl3)
δ −118.85 (ddd, J = 26.3, 15.9, 10.3 Hz), −121.79 (p, J = 12.1 Hz), −126.42. HRMS
(ESI): m/z [M+H]+ calcd. for C32H31F6N3O3Si: 648.2117, found: 648.2110.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
9-(4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)-9H-purin-6-amine
(18a)
Compound 18a was prepared from 14a using the same procedure as for compound
1
15a; yield 92%. H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.91 (d, J = 1.0 Hz,
1H), 7.80 – 7.37 (m, 10H), 5.87 (s, 2H), 4.95 (t, J = 16.0 Hz, 2H), 4.09 (tt, J = 13.1,
1.6 Hz, 2H), 1.11 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 155.55, 153.64, 150.85,
140.78, 135.56, 131.69, 130.26, 128.02, 26.60, 19.25. 19F NMR (377 MHz, CDCl3)
δ −119.58 (t, J = 16.0 Hz), −123.18 (t, J = 13.1 Hz). HRMS (ESI): m/z [M+H]+
calcd. for C25H27F4N5OSi: 518.1999, found: 518.1996.
9-(5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)-9H-purin-6-
amine (18b)
Compound 18b was prepared from 14b using the same procedure as for compound
1
15a; yield 80%. H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.90 (s, 1H), 7.75 –
7.37 (m, 10H), 7.07 (s, 2H), 4.90 (t, J = 15.8 Hz, 2H), 4.20 – 4.04 (m, 2H), 1.11 (s,
9H). 13C NMR (101 MHz, CDCl3) δ 156.23, 153.67, 150.61, 140.36, 135.56, 131.90,
130.21, 127.98, 118.89, 61.81 (t, J = 27.0 Hz), 42.85 (t, J = 22.9 Hz), 26.50, 19.30. 19F
NMR (377 MHz, CDCl3) δ −118.38 (p, J = 14.3, 13.0 Hz), −121.69 (p, J = 12.3 Hz),
−126.09. HRMS (ESI): m/z [M+H]+ calcd. for C26H27F6N5OSi: 568.1967, found:
568.1960.
9-(4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)-6-chloro-9H-purin-
2-amine (19a)
Compound 19a was prepared from 14a using the same procedure as for compound
15a; yield 80%. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.72 (dt, J = 6.6, 1.6 Hz,
4H), 7.47 (ddd, J = 14.1, 7.7, 6.1 Hz, 6H), 5.30 (s, 2H), 4.86 (t, J = 16.1 Hz, 2H), 4.19
– 4.00 (m, 2H), 1.12 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 159.49, 154.52, 151.66,
142.28, 135.54, 131.60, 130.36, 128.07, 124.60, 61.60 (t, J = 29.5 Hz), 43.03 (t, J =
23.1 Hz), 26.64, 19.25. 19F NMR (377 MHz, CDCl3) δ −119.59 (t, J = 16.1 Hz),
−123.25 (t, J = 12.6 Hz). HRMS (ESI): m/z [M+H]+ calcd. for C25H26ClF4N5OSi:
552.1610, found: 552.1608.
9-(5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)-6-chloro-9H-
purin-2-amine (19b)
Compound 19b was prepared from 14b using the same procedure as for compound
15a; yield 63%. 1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.75 – 7.39 (m, 10H),
5.65 (s, 2H), 4.75 (t, J = 15.7 Hz, 2H), 4.11 (t, J = 13.6 Hz, 2H), 1.11 (s, 9H). 13C NMR
(101 MHz, CDCl3) δ 159.71, 154.43, 151.70, 142.01, 135.54, 131.85, 130.24, 127.99,
124.42, 118.43 – 110.55 (m), 61.80 (t, J = 27.2 Hz), 42.83 (t, J = 23.2 Hz), 26.49, 19.30.
19F NMR (377 MHz, CDCl3) δ −118.35 (ddd, J = 26.5, 16.6, 10.6 Hz), −121.64 (p,
J = 11.3 Hz), −126.14. HRMS (ESI): m/z [M+H]+ calcd. for C26H26ClF6N5OSi:
602.1578, found: 602.1574.

10
S. MAHMOUD ET AL.
1-(4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)pyrimidine-
2,4(1H,3H)-dione (20a)
Compound 15a (0.51 mmol, 307 mg) was dissolved in saturated NH3 in CH3OH
(15 mL) and stirred at room temperature overnight. The reaction mixture was con-
centrated, extracted with ethyl acetate (3 × 10 mL), dried over Na2SO3 and purified
by column chromatography on silica gel, eluting with dichloromethane/methanol
1
(92:8) to afford compound 20a (0.41 mmol, 203 mg, 80%) as white powder. H
NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 7.77 – 7.34 (m, 10H), 7.22 (dd, J = 8.1,
1.2 Hz, 1H), 5.81 (dd, J = 8.1, 2.0 Hz, 1H), 4.52 (t, J = 15.5 Hz, 2H), 4.05 (td, J =
13.0, 1.9 Hz, 2H), 1.10 (d, J = 2.2 Hz, 9H). 13C NMR (101 MHz, CDCl3) δ 163.13,
150.57, 144.24, 135.54, 131.65, 130.27, 128.04, 103.15, 61.37, 26.59, 19.24. 19F NMR
(377 MHz, CDCl3) δ −120.23 (t, J = 15.7 Hz), −123.56 (t, J = 13.0 Hz). HRMS
(ESI): m/z [M+H]+ calcd. for C24H26F4N2O3Si: 493.1571, found: 493.1581.
1-(5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)pyrimidine-
2,4(1H,3H)-dione (20b)
Compound 20b was prepared from 15b using the same procedure as for compound
20a; yield 78%. 1H NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 7.79 – 7.38 (m, 10H),
7.25 (d, J = 8.0 Hz, 1H), 5.85 (d, J = 8.0 Hz, 1H), 4.50 (t, J = 15.5 Hz, 2H), 4.12
(t, J = 13.7 Hz, 2H), 1.13 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 164.00, 150.91,
144.28, 135.57, 131.94, 130.23, 128.01, 119.22 – 107.63 (m), 103.38, 61.81 (t, J =
26.9 Hz), 46.19 (t, J = 21.7 Hz), 26.52, 19.33. 19F NMR (377 MHz, CDCl3) δ −118.94
(p, J = 14.2 Hz), −121.74 (p, J = 12.2 Hz), −126.48. HRMS (ESI): m/z [M+H]+
calcd. for C25H26F6N2O3Si: 545.1695, found: 545.1686.
1-(4-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)-5-methylpyrimidine-
2,4(1H,3H)-dione (21a)
Compound 21a was prepared from 16a using the same procedure as for compound
20a; yield 90%. 1H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 7.77 – 7.38 (m, 10H),
7.05 (s, 1H), 4.49 (t, J = 15.6 Hz, 2H), 4.05 (t, J = 13.0 Hz, 2H), 1.97 (d, J = 1.3 Hz,
3H), 1.11 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 163.83, 150.76, 140.03, 135.55,
131.70, 130.26, 128.03, 111.60, 61.38 (t, J = 29.0 Hz), 46.20 (t, J = 22.0 Hz), 26.60,
19.25, 12.36. 19F NMR (377 MHz, CDCl3) δ −120.18 (t, J = 15.7 Hz), −123.63 (t, J =
13.1 Hz). HRMS (ESI): m/z [M+H]+ calcd. for C25H28F4N2O3Si: 509.1884, found:
509.1877.
1-(5-((Tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (21b)
Compound 21b was prepared from 16b using the same procedure as for compound
20a; yield 87%. 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 7.91 – 7.30 (m, 10H),
7.08 – 7.04 (m, 1H), 4.45 (t, J = 15.6 Hz, 2H), 4.11 (t, J = 13.7 Hz, 2H), 2.16 – 1.81
(m, 3H), 1.12 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 164.38, 151.05, 139.81, 135.56,
131.94, 130.21, 127.98, 111.93, 61.80 (t, J = 27.1 Hz), 46.04 (t, J = 21.8 Hz), 26.51,
19.32, 12.31. 19F NMR (377 MHz, CDCl3) δ −118.90 (p, J = 12.9, 12.4 Hz), −121.79

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
(p, J = 12.1 Hz), −126.55. HRMS (ESI): m/z [M+H]+ calcd. for C26H28F6N2O3Si:
559.1852, found: 559.1844.
4-Amino-1-(4-((tert-butyldiphenylsilyl)oxy)-2,2,3,3-tetrafluorobutyl)pyrimidin-
2(1H)-one (22a)
Compound 22a was prepared from 17a using the same procedure as for compound
1
20a; yield 52%. H NMR (400 MHz, CDCl3) δ 7.80 – 7.28 (m, 11H), 5.74 (dd,
J = 7.2, 1.3 Hz, 1H), 4.58 (t, J = 15.9 Hz, 2H), 4.05 (t, J = 13.3 Hz, 2H), 1.09 (s,
9H). 13C NMR (101 MHz, CDCl3) δ 165.50, 155.88, 146.20, 135.56, 131.87, 130.16,
127.97, 94.59, 26.58, 19.26.19F NMR (377 MHz, CDCl3) δ −120.11 (t, J = 15.9 Hz),
−123.57 (t, J = 13.1 Hz). HRMS (ESI): m/z [M+H]+ calcd. for C24H27F4N3O2Si:
494.1887, found: 494.1884.
4-Amino-1-(5-((tert-butyldiphenylsilyl)oxy)-2,2,3,3,4,4-hexafluoropentyl)
pyrimidin-2(1H)-one (22b)
Compound 22b was prepared from 17b using the same procedure as for com-
pound 20a; yield 50%. 1H NMR (400 MHz, CDCl3) δ 7.71 – 7.26 (m, 10H), 7.19 (d,
J = 7.4 Hz, 1H), 5.83 (d, J = 7.3 Hz, 1H), 4.49 (t, J = 16.0 Hz, 2H), 4.06 (t, J =
13.7 Hz, 2H), 1.09 (s, 9H). 13C NMR (101 MHz,CDCl3) δ 166.08, 156.22, 145.18,
135.53, 131.97, 130.14, 127.93, 95.79, 61.77 (t, J = 25.7 Hz), 47.20 – 46.72 (m), 26.49,
19.30. 19F NMR (377 MHz, CDCl3) δ −119.17 (dq, J = 16.9, 11.1, 8.9 Hz), −121.94
(p, J = 12.3 Hz), −126.66. HRMS (ESI): m/z [M+H]+ calcd. for C25H27F6N3O2Si:
544.1855, found: 544.1854.
1-(2,2,3,3-Tetrafluoro-4-hydroxybutyl)pyrimidine-2,4(1H,3H)-dione (23a)
To a solution of compound 20a (0.68 mmol, 338 mg) in THF (8 mL) was
added tBuNH4+F−in THF (1.02 mmol, 1 mL, 1.5 eq). The reaction mixture was
stirred for 4 hours at room temperature and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography eluting with
dichloromethane/methanol (95:5) to afford compound 23a (0.66 mmol, 169 mg,
1
97%) as a white powder. H NMR (400 MHz, DMSO) δ 11.50 (s, 1H), 7.62 (d,
J = 7.9 Hz, 1H), 6.03 (t, J = 6.3 Hz, 1H), 5.66 (d, J = 7.9 Hz, 1H), 4.51 (t, J = 16.7 Hz,
2H), 3.91 (td, J = 14.8, 6.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 163.83, 151.35,
146.21, 102.17, 59.54 – 58.73 (m), 46.36 – 45.63 (m). 19F NMR (377 MHz, DMSO)
δ −120.44 (t, J = 16.8 Hz), −124.28 (t, J = 14.8 Hz). HRMS (ESI): m/z [M+H]+
calcd. for C8H8F4N2O3: 257.0549, found: 257.0542.
1-(2,2,3,3,4,4-Hexafluoro-5-hydroxypentyl)pyrimidine-2,4(1H,3H)-dione (23b)
Compound 23b was prepared from 20b using the same procedure as for compound
1
23a; yield 80%. H NMR (400 MHz, MeOD) δ 7.60 (d, J = 7.9 Hz, 1H), 5.75 (d,
J = 7.8 Hz, 1H), 4.60 (t, J = 16.1 Hz, 2H), 4.03 (t, J = 15.0 Hz, 2H). 13C NMR
(101 MHz, MeOD) δ 164.72, 151.17, 145.52, 101.81, 59.31 (t, J = 26.1 Hz), 45.62.

12
S. MAHMOUD ET AL.
19F NMR (377 MHz, MeOD) δ −120.35 (tt, J = 16.9, 9.7 Hz), −124.21 to −124.51
(m), −128.87. HRMS (ESI): m/z [M+H]+ calcd. for C9H8F6N2O3: 307.0517, found:
307.0511.
5-Methyl-1-(2,2,3,3-tetrafluoro-4-hydroxybutyl)pyrimidine-2,4(1H,3H)-dione
(24a)
Compound 24a was prepared from 21a using the same procedure as for compound
23a; yield 97%. 1H NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 7.53 − 7.49 (m, 1H),
6.02 (t, J = 6.4 Hz, 1H), 4.46 (t, J = 16.7 Hz, 2H), 3.91 (td, J = 14.8, 6.0 Hz, 2H), 1.77
(d, J = 1.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 164.48, 151.39, 141.76, 109.71,
59.04 (t, J = 26.3 Hz), 45.88 (t, J = 21.0 Hz), 12.34. 19F NMR (377 MHz, DMSO)
δ −120.36 (t, J = 16.7 Hz), −124.33 (t, J = 14.9 Hz). HRMS (ESI): m/z [M+H]+
calcd. for C9H10F4N2O3: 271.0706, found: 271.0700.
1-(2,2,3,3,4,4-Hexafluoro-5-hydroxypentyl)-5-methylpyrimidine-2,4(1H,3H)-
dione (24b)
Compound 24b was prepared from 21b using the same procedure as for compound
23a; yield 73%. 1H NMR (400 MHz, MeOD) δ 7.43 (s, 1H), 4.56 (t, J = 16.5 Hz, 2H),
4.03 (t, J = 15.1 Hz, 2H), 1.90 (d, J = 1.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ
165.09, 151.45, 141.16, 119.67 – 111.11 (m), 110.72, 59.30 (t, J = 25.2 Hz), 45.55 (t,
J = 21.4 Hz), 10.81. 19F NMR (377 MHz, MeOD) δ −120.25 (tt, J = 17.6, 9.7 Hz),
−124.30 (ddd, J = 24.9, 15.5, 9.6 Hz), −128.80. HRMS (ESI): m/z [M+H]+ calcd.
for C10H10F6N2O3: 321.0674, found: 321.0667.
4-Amino-1-(2,2,3,3-tetrafluoro-4-hydroxybutyl)pyrimidin-2(1H)-one(25a)
Compound 25a was prepared from 22a using the same procedure as for compound
1
23a; yield 80%. H NMR (400 MHz, DMSO) δ 7.52 (d, J = 7.3 Hz, 1H), 7.24 (d,
J = 37.7 Hz, 2H), 6.00 (s, 1H), 5.72 (d, J = 7.3 Hz, 1H), 4.49 (t, J = 16.9 Hz, 2H),
3.90 (t, J = 15.1 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 166.48, 155.95, 146.73,
94.70, 59.49 – 58.63 (m), 46.81 – 46.07 (m). 19F NMR (377 MHz, DMSO) δ −120.28
(t, J = 16.9 Hz), −124.36 (t, J = 14.9 Hz). HRMS (ESI): m/z [M+H]+ calcd. for
C8H9F4N3O2: 256.0709, found: 256.0702.
4-Amino-1-(2,2,3,3,4,4-hexafluoro-5-hydroxypentyl)pyrimidin-2(1H)-one(25b)
Compound 25b was prepared from 22b using the same procedure as for compound
23a; yield 79%. 1H NMR (400 MHz, MeOD) δ 7.58 (d, J = 7.4 Hz, 1H), 5.93 (d, J =
7.4 Hz, 1H), 4.64 (t, J = 16.4 Hz, 2H), 4.02 (t, J = 15.1 Hz, 2H). 13C NMR (101 MHz,
MeOD) δ 166.64, 157.17, 146.10, 95.19, 59.34 (t, J = 25.0 Hz), 46.76 – 46.23 (m). 19F
NMR (377 MHz, MeOD) δ −120.40 (tt, J = 16.9, 9.7 Hz), −124.45 (q, J = 12.2,
9.2 Hz), −128.92. HRMS (ESI): m/z [M+H]+ calcd. for C9H9F6N3O2: 306.0677,
found: 306.0669.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
4-(6-Amino-9H-purin-9-yl)-2,2,3,3-tetrafluorobutan-1-ol(26a)
Compound 26a was prepared from 18a using the same procedure as for com-
1
pound 23a; yield 89%. H NMR (400 MHz, DMSO) δ 8.19 (d, J = 6.1 Hz, 2H),
7.38 (s, 2H), 6.13 (s, 1H), 4.98 (t, J = 17.0 Hz, 2H), 3.99 (td, J = 14.7, 6.2 Hz,
2H). 13C NMR (101 MHz, DMSO) δ 156.53, 153.45, 150.60, 141.83, 118.50, 59.16
(t, J = 26.3 Hz), 42.90 (t, J = 22.7 Hz). 19F NMR (377 MHz, DMSO) δ −119.73
(t, J = 17.2 Hz), −123.94 (t, J = 14.7 Hz). HRMS (ESI): m/z [M+H]+ calcd. for
C9H9F4N5O: 280.0821, found: 280.0813.
5-(6-Amino-9H-purin-9-yl)-2,2,3,3,4,4-hexafluoropentan-1-ol (26b)
Compound 26b was prepared from 18b using the same procedure as for compound
23a; yield 70%. 1H NMR (400 MHz, DMSO) δ 8.24 – 8.13 (m, 2H), 7.38 (s, 2H),
6.04 (t, J = 6.5 Hz, 1H), 5.05 (t, J = 16.8 Hz, 2H), 4.06 – 3.91 (m, 2H). 13C NMR
(101 MHz, CDCl3) δ 160.02, 156.87, 153.98, 145.63, 122.04, 63.54 – 62.88 (m),
46.39 (t, J = 23.8 Hz). 19F NMR (377 MHz, DMSO) δ −117.94 (dq, J = 16.8, 9.2,
8.6 Hz), −121.83 to −122.23 (m), −126.56. HRMS (ESI): m/z [M+H]+ calcd. for
C10H9F6N5O: 330.0790, found: 330.0782.
2-Amino-9-(2,2,3,3-tetrafluoro-4-hydroxybutyl)-1,9-dihydro-6H-purin-6-one
(27a)
A solution of compound 19a (0.21 mmol, 120 mg) in 80% acetic acid was
stirred at 110°C overnight (28 mL) and evaporated under reduced pressure.
The residue was purified by column chromatography on silica gel, eluting with
dichloromethane/methanol (85:15) to afford compound 27a (0.09 mmol, 25 mg,
43%) as a yellow solid; 1H NMR (400 MHz, DMSO) δ 10.72 (s, 1H), 7.72 (s, 1H),
6.59 (s, 2H), 6.09 (t, J = 6.4 Hz, 1H), 4.74 (t, J = 17.1 Hz, 2H), 3.95 (td, J = 14.7,
5.1 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 157.23, 154.36, 152.42, 138.46, 116.38,
59.06 (t, J = 25.8 Hz), 42.57 (t, J = 22.1 Hz). 19F NMR (377 MHz, DMSO) δ −119.87
(t, J = 17.1 Hz), −123.88 (t, J = 15.1 Hz). HRMS (ESI): m/z [M+H]+ calcd. for
C9H9F4N5O2: 296.0771, found: 296.0764.
2-Amino-9-(2,2,3,3,4,4-hexafluoro-5-hydroxypentyl)-1,9-dihydro-6H-purin-6-
one (27b)
Compound 27b was prepared from 19busing the same procedure as for com-
1
pound27a; yield 40%. H NMR (400 MHz, DMSO) δ 10.74 (s, 1H), 7.76 (s, 1H),
6.62 (s, 2H), 6.04 (t, J = 6.5 Hz, 1H), 4.83 (t, J = 17.1 Hz, 2H), 3.98 (td, J =
15.7, 5.6 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 157.19, 154.45, 152.43, 138.36,
116.42, 59.29 (t, J = 24.7 Hz), 42.22 (t, J = 22.0 Hz). 19F NMR (377 MHz,
DMSO) δ −118.14 (tt, J = 18.3, 10.0 Hz), −121.96 (tt, J = 17.1, 9.9 Hz), −126.45.
HRMS (ESI): m/z [M+H]+ calcd. for C10H9F6N5O2: 346.0739, found: 346.
0733.

14
S. MAHMOUD ET AL.
Ethyl((4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,3,3-tetrafluorobutoxy)
(phenoxy)phosphoryl)-L-alaninate(29)
To a solution of derivative 23a (0.16 mmol, 41 mg) in dry THF (2 mL) was added
t-BuMgCl (0.40 mmol, 0.4 mL, 2.5 eq, 1 M in THF) at 0°C. After 10 minutes, a
solution of ethyl (chloro(phenoxy)phosphoryl)-L-alaninatereagent 28 (0.40 mmol,
117 mg, 2.5 eq.) in dry THF (1 mL) was added at 0°C and the mixture was stirred
for 3 hours, concentrated under reduced pressure, and purified by silica gel column
chromatography eluting with dichloromethane/methanol (96:4) to afford prodrug
29 (0.09 mmol, 46 mg, 56%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.76 (s,
1H), 7.42 – 7.14 (m, 6H), 5.79 (dd, J = 8.0, 3.0 Hz, 1H), 4.69 – 3.86 (m, 8H), 1.41 (t,
J = 7.5 Hz, 3H), 1.28 (q, J = 6.9 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −119.69
(q, J = 14.4 Hz), −123.11 (q, J = 13.1, 12.6 Hz). 31P NMR (162 MHz, CDCl3) δ
2.28, 2.06. HRMS (ESI): m/z [M+H]+ calcd. for C19H22F4N3O7P: 512.1210, found:
512.1202.
Ethyl(phenoxy(2,2,3,3-tetrafluoro-4-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)butoxy)phosphoryl)-L-alaninate (30)
Title compound 30 was prepared from 24a using the same procedure as for com-
pound 29; yield 60%. 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.40 – 7.15 (m,
5H), 7.00 (d, J = 7.8 Hz, 1H), 4.68 – 3.84 (m, 8H), 1.94 (t, J = 1.5 Hz, 3H), 1.39 (dd,
J = 9.0, 7.0 Hz, 3H), 1.31 – 1.21 (m, 3H). 19F NMR (377 MHz, CDCl3) δ −119.59 (td,
J = 19.9, 19.4, 12.9 Hz), −123.16 (q, J = 12.0, 10.8 Hz). 31P NMR (162 MHz, CDCl3)
δ 2.29, 2.06. HRMS (ESI): m/z [M+H]+ calcd. for C20H24F4N3O7P: 526.1366,
found: 526.1360.
Ethyl((4-(4-amino-2-oxopyrimidin-1(2H)-yl)-2,2,3,3-tetrafluorobutoxy)
(phenoxy)phosphoryl)-L-alaninate (31)
Compound 31 was prepared from 25a using the same procedure as for compound
29; yield 35%. 1H NMR (400 MHz, CDCl3) δ 7.39 – 7.14 (m, 6H), 5.80 (d, J = 7.2 Hz,
1H), 4.82 – 3.99 (m, 8H), 1.40 (dd, J = 14.6, 7.0 Hz, 3H), 1.27 (td, J = 7.1, 4.5 Hz,
3H). 19F NMR (377 MHz, CDCl3) δ −119.27 to −119.80 (m), −123.21 to −123.37
(m). 31P NMR (162 MHz, CDCl3) δ 2.74, 2.36.HRMS (ESI): m/z [M+H]+ calcd. for
C19H23F4N4O6P: 511.1370, found: 511.1363.
Ethyl((4-(6-amino-9H-purin-9-yl)-2,2,3,3tetrafluorobutoxy)(phenoxy)
phosphoryl)-L-alaninate (32)
To a solution of 26a (0.18 mmol, 50 mg) in THF (2 mL) at 0°C was added NMI
(0.72 mmol, 57 mL, 4 eq.). The reaction was stirred at 0°C for 15 minutes. Then, ethyl
(chloro(phenoxy)phosphoryl)-L-alaninate28 (0.36 mmol, 105 mg, 2 eq.) was slowly
added. The mixture was stirred for 2 hours at room temperature and quenched
with ethyl acetate (4 mL). The organic layer was washed with water and dried over

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Na2SO3. After concentrated under reduced pressure was purified by silica gel col-
umn chromatography eluting with dichloromethane/methanol (96:4) to afford pro-
drug 32 (0.12 mmol, 58 mg, 60%) as a white solid; 1H NMR (400 MHz, CDCl3) δ
8.38 (d, J = 30.4 Hz, 1H), 7.87 (d, J = 6.4 Hz, 1H), 7.37 – 7.12 (m, 5H), 6.20 (d, J =
4.1 Hz, 2H), 4.99 – 4.44 (m, 5H), 4.23 – 4.02 (m, 3H), 1.40 (t, J = 6.8 Hz, 3H), 1.24
(td, J = 7.2, 3.0 Hz, 3H). 19F NMR (377 MHz, CDCl3) δ −119.22 (t, J = 16.3 Hz),
−122.72 (dt, J = 26.1, 13.5 Hz). 31P NMR (162 MHz, CDCl3) δ 2.52, 2.36. HRMS
(ESI): m/z [M+H]+ calcd. for C20H23F4N6O5P: 535.1482, found: 535.1472.
Ethyl((4-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2,3,3-tetrafluorobutoxy)
(phenoxy)phosphoryl)-L-alaninate (33)
To a solution of 27a (0.13 mmol, 38 mg) in THF (4 mL) and DMF (1 mL) at 0°C
was added tBuMgCl (2 M in ether, 0.26 mmol, 0.26 mL, 2 eq.). The reaction was
stirred for 15 minutes. Then, ethyl (chloro(phenoxy)phosphoryl)-L-alaninate 28
(0.26 mmol, 76 mg, 2 eq.) was slowly added at 0°C. The reaction mixture was stirred
for 3 hours at room temperature and quenched with a saturated solution of NH4Cl
and extracted with CH2Cl2. The combined organic layers were dried over Na2SO3,
concentrated under reduced pressure, and purified by silica gel column chromatog-
raphy dichloromethane/methanol (86:14) to afford 33 (0.07 mmol, 39 mg, 56%) as
a yellow solid; 1H NMR (400 MHz, DMSO) δ 10.66 (d, J = 10.5 Hz, 1H), 7.70 (d,
J = 3.9 Hz, 1H), 7.49 – 7.11 (m, 5H), 6.57 (s, 2H), 6.33 (ddd, J = 15.2, 13.3, 9.9 Hz,
1H), 4.87 – 4.49 (m, 2H), 4.15 – 3.96 (m, 5H), 1.23 (t, J = 6.3 Hz, 3H), 1.19 – 1.09
(m, 3H). 19F NMR (377 MHz, DMSO) δ −119.10 to −119.47 (m), −122.95 (dt, J =
45.6, 14.7 Hz). 31P NMR (162 MHz, DMSO) δ 3.66, 3.25. HRMS (ESI): m/z [M+H]+
calcd. for C20H23F4N6O6P: 551.1431, found: 551.1428.
Isopropyl(((5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,3,3,4,4-
hexafluoropentyl)oxy)(phenoxy)phosphoryl)-L-alaninate (35)
Compound 35 was obtained from 23b using the same procedure as for compound
29 using reagent 34 instead of 28; yield 48%. 1H NMR (400 MHz, CDCl3) δ 8.99
(s, 1H), 7.46 – 7.11 (m, 6H), 5.97 – 5.69 (m, 1H), 5.19 – 4.96 (m, 1H), 4.68 – 4.29
(m, 4H), 3.99 (dd, J = 10.2, 3.5 Hz, 2H), 1.40 (d, J = 4.6 Hz, 3H), 1.26 (dd, J = 6.2,
3.1 Hz, 6H). 19F NMR (377 MHz, CDCl3) δ −118.84 (p, J = 11.7 Hz), −121.54 (t,
J = 12.9 Hz), −126.95 (d, J = 11.8 Hz). 31P NMR (162 MHz, CDCl3) δ 2.18, 2.07.
HRMS (ESI): m/z [M+H]+ calcd. for C21H24F6N3O7P: 576.1334, found: 576.1330.
Isopropyl(((2,2,3,3,4,4-hexafluoro-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)pentyl)oxy)(phenoxy)phosphoryl)-L-alaninate (36)
Compound 36 was obtained from 24b using the same procedure as for compound
29 using reagent 34 instead of 28; yield 45%. 1H NMR (400 MHz, MeOD) δ 7.53
– 7.16 (m, 6H), 5.00 (pd, J = 6.2, 1.5 Hz, 1H), 4.61 (dddd, J = 32.0, 15.9, 11.2,

16
S. MAHMOUD ET AL.
6.0 Hz, 4H), 3.93 (dq, J = 9.5, 7.1 Hz, 1H), 1.90 (d, J = 1.4 Hz, 3H), 1.39 – 1.33
(m, 3H), 1.25 (dq, J = 6.4, 2.0 Hz, 6H). 19F NMR (377 MHz, MeOD) δ −120.03
(td, J = 16.6, 16.2, 8.3 Hz), −122.77 (p, J = 13.0 Hz), −128.26 (d, J = 22.6 Hz).
31P NMR (162 MHz, MeOD) δ 3.36, 3.01. HRMS (ESI): m/z [M+H]+ calcd. for
C22H26F6N3O7P: 590.1491, found: 590.1483.
Isopropyl(((5-(4-amino-2-oxopyrimidin-1(2H)-yl)-2,2,3,3,4,4-
hexafluoropentyl)oxy) (phenoxy)phosphoryl)-L-alaninate (37)
Compound 37 was obtained from 25b using the same procedure as for compound
29 using reagent 34 instead of 28; yield 39%. 1H NMR (400 MHz, CDCl3) δ 8.28 (s,
1H), 7.46 – 7.08 (m, 6H), 6.75 (s, 1H), 5.93 (dd, J = 7.4, 3.5 Hz, 1H), 5.08 – 4.95 (m,
1H), 4.51 (dddd, J = 32.7, 20.5, 14.4, 5.7 Hz, 4H), 4.17 (dd, J = 11.9, 9.4 Hz, 1H),
3.99 (dtd, J = 13.0, 6.7, 6.2, 3.3 Hz, 1H), 1.40 (d, J = 7.0 Hz, 3H), 1.24 (td, J = 6.1,
5.6, 3.1 Hz, 6H). 19F NMR (377 MHz, CDCl3) δ −118.90 to −119.26 (m), −121.72
(dt, J = 22.4, 10.3 Hz), −127.43, −127.61. 31P NMR (162 MHz, CDCl3) δ 2.34, 2.30.
HRMS (ESI): m/z [M+H]+ calcd. for C21H25F6N4O6P: 575.1494, found: 575.1488.
Isopropyl(((5-(6-amino-9H-purin-9-yl)-2,2,3,3,4,4-hexafluoropentyl)oxy)
(phenoxy)phosphoryl)-L-alaninate (38)
Compound 38 was obtained from 26b using the same procedure as for compound
32 using reagent 34 instead of 28; yield 60%. 1H NMR (400 MHz, CDCl3) δ 8.40 (d,
J = 9.2 Hz, 1H), 7.90 (d, J = 3.0 Hz, 1H), 7.38 – 7.17 (m, 5H), 6.05 (s, 2H), 5.08 –
3.91 (m, 7H), 1.40 (d, J = 7.0 Hz, 3H), 1.27 – 1.21 (m, 6H). 19F NMR (377 MHz,
CDCl3) δ −118.20 to −118.43 (m), −121.54 (p, J = 12.9 Hz), −126.63, −126.83 (d,
J = 8.4 Hz). 31P NMR (162 MHz, CDCl3) δ 2.39, 2.27. HRMS (ESI): m/z [M+H]+
calcd. for C22H25F6N6O5P: 599.1606, found: 599.1596.
Isopropyl(((5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,2,3,3,4,4-
hexafluoropentyl)oxy)(phenoxy)phosphoryl)-L-alaninate (39)
Compound 39 was obtained from 27b using the same procedure as for compound
33 using reagent 34 instead of 28; yield 50%. 1H NMR (400 MHz, DMSO) δ 10.74
(s, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.47 – 7.13 (m, 5H), 6.59 (s, 2H), 6.32 (td, J =
14.2, 13.5, 9.9 Hz, 1H), 4.92 – 4.54 (m, 6H), 1.22 (dd, J = 6.9, 4.6 Hz, 3H), 1.16
– 1.13 (m, 6H). 19F NMR (377 MHz, DMSO) δ −117.97 (tt, J = 18.0, 8.9 Hz),
−121.03 (dq, J = 24.4, 12.8, 11.9 Hz), −126.22. 31P NMR (162 MHz, DMSO) δ 3.45,
3.04. HRMS (ESI): m/z [M+H]+ calcd. for C22H25F6N6O6P: 615.1556, found: 615.
1545.
Acknowledgments
We wish to thank Mervi Detorio and Cathy Montero for excellent technical assistance.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
Funding
This work was supported in part by NIH grant 5P30-AI-50409 (CFAR). We would like to
acknowledge the Egyptian Ministry of Higher Education and the Egyptian Educational Culture
Bureau in USA for funding Dr. Sawsan’s Ph.D. studies at Emory University. Dr. Schinazi is the
Chairman and a major shareholder of CocrystalPharma, Inc. Emory received no funding from
CocrystalPharma, Inc. to perform this work and vice versa.
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