Synthesis of new 2′-deoxy-2′-fluoro-4′-azido nucleoside analogues as potent anti-HIV agents

Article abstract of DOI:10.1016/j.ejmech.2011.06.020

We prepared 1-(4′-azido-2′-deoxy-2′-fluoro-β-d- arabinofuranosyl)cytosine (10) and its hydrochloride salt (11) as potential antiviral agents based on the favorable antiviral profiles of 4′-substituted nucleosides. Compounds 10 and 11 were synthesized from 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-d-arabinofuranoside in multiple steps, and their structures were unequivocally established by IR, ¹H NMR, ¹³C NMR, and ¹⁹F NMR spectroscopy, HRMS, and X-ray crystallography. Compounds 10 and 11 exhibited potent anti-HIV-1 activity (EC₅₀: 0.3 and 0.13 nM, respectively) without significant cytotoxicity in concentrations up to 100 μM. Compound 11 exhibited extremely potent anti-HIV activity against NL4-3 (wild-type), NL4-3 (K101E), and RTMDR viral strains, with EC₅₀ values of 0.086, 0.15, and 0.11 nM, respectively. Due to the high potency of 11, it was also screened against an NIH Reagent Program NRTI-resistant virus panel containing eleven mutated viral strains and for cytotoxicity against six different human cell lines. The results of this screening indicated that 11 is a novel NRTI that could be developed as an anti-AIDS clinical trial candidate to overcome drug-resistance issues.

Full text of DOI:10.1016/j.ejmech.2011.06.020

European Journal of Medicinal Chemistry 46 (2011) 4178e4183
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new 20-deoxy-20-fluoro-40-azido nucleoside analogues as potent
anti-HIV agents
Qiang Wang a, Weidong Hu a, Shuyang Wang a, Zhenliang Pan a, Le Tao b, Xiaohe Guo b, Keduo Qian c,
,
e
,
a,
**
Chin-Ho Chen d, Kuo-Hsiung Lee c , Junbiao Chang
*
a Department of Chemistry, Zhengzhou University, Zhengzhou 450001, PR China
b Hena Key Laboratory of Fine Chemical, Zhengzhou 450002, PR China
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
d Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
We prepared 1-(40-azido-20-deoxy-20-fluoro-
b-D-arabinofuranosyl)cytosine (10) and its hydrochloride
Article history:
salt (11) as potential antiviral agents based on the favorable antiviral profiles of 40-substituted nucleo-
Received 1 March 2011
Received in revised form
13 June 2011
Accepted 16 June 2011
Available online 23 June 2011
sides. Compounds 10 and 11 were synthesized from 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-D-arabinofur-
anoside in multiple steps, and their structures were unequivocally established by IR, 1H NMR, 13C NMR,
and 19F NMR spectroscopy, HRMS, and X-ray crystallography. Compounds 10 and 11 exhibited potent
anti-HIV-1 activity (EC50: 0.3 and 0.13 nM, respectively) without significant cytotoxicity in concentra-
tions up to 100 mM. Compound 11 exhibited extremely potent anti-HIV activity against NL4-3 (wild-
Keywords:
type), NL4-3 (K101E), and RTMDR viral strains, with EC50 values of 0.086, 0.15, and 0.11 nM, respectively.
Due to the high potency of 11, it was also screened against an NIH Reagent Program NRTI-resistant virus
panel containing eleven mutated viral strains and for cytotoxicity against six different human cell lines.
The results of this screening indicated that 11 is a novel NRTI that could be developed as an anti-AIDS
clinical trial candidate to overcome drug-resistance issues.
40-Azido-20-deoxy-20-fluoro nucleosides
Anti-HIV activity
Nucleoside reverse transcriptase inhibitor
(NRTI)
Drug-resistance
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
worldwide in 2007, 2.1 million deaths from AIDS, and 2.5 million
newly infected patients with AIDS. Over the last two decades,
Since the discovery of the human immunodeficiency virus (HIV)
in 1983, AIDS has become the leading infectious cause of death
worldwide. According to the World Health Organization (07 AIDS
Epidemic Update), there were 33.2 million people living with AIDS
tremendous effort has been directed to the discovery and devel-
opment of novel agents for the treatment of HIV infections and
great successes have been achieved. So far, there are more than 20
approved anti-HIV drugs, belonging to seven classes [1].
Nucleoside reverse transcriptase inhibitors (NRTIs) are one of
the most important classes of compounds active against HIV
replication and have been used extensively to treat HIV infection
[2]. However, the long term use of antiviral nucleoside analogs is
inevitably associated with delayed toxicity and/or development of
drug-resistant virus [3e6], which limits their effectiveness as an
AIDS treatment. Therefore, there remains a need for the develop-
ment of novel anti-HIV agents with better therapeutic indices, new
mechanisms of action, and activity against HIV strains resistant to
currently available drugs.
Abbreviations: IR, infrared; NMR, nuclear magnetic resonance; HRMS, high
resolution mass spectrometry; RT, reverse transcriptase; RTMDR, reverse tran-
scriptase multi-drug resistant; NRTI, nucleoside reverse transcriptase inhibitor;
NNRTI, non-nucleoside reverse transcriptase inhibitor; HIV, human immunodefi-
ciency virus; AIDS, acquired immunodeficiency syndrome; dN, 20-deoxynucleoside;
ddN, 20,30-dideoxynucleoside; 40sdN, 40-C-substituted-20-deoxynucleoside; DCM,
dichloromethane; THF, tetrahydrofuran; m-CPBA, m-chloroperbenzoic acid; m-CBA,
m-chlorobenzoic acid; EFV, efavirenz; AZT, zidovudine; HMDS, hexamethyldisila-
zane; VSVG, vesicular stomatitis virus glycoprotein.
* Corresponding author. Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA.
Tel.: þ1 919 962 0066; fax: þ1 919 966 3893.
All clinical NRTIs belong to the family of 20,30-dideoxynucleoside
(ddN) [7]. As the chain terminator of proviral DNA biosynthesis, the
ddN structure has been assumed to be essential for an active
nucleoside derivative. However, resistant HIV variants and other
side effects have emerged. Resistance to these ddNs results from
** Corresponding author. No. 100, Science Road, Zhengzhou University, Zhengz-
hou, Henan, PR China. Tel./fax: þ86 371 67783017.
E-mail addresses: khlee@unc.edu (K.-H. Lee), changjunbiao@zzu.edu.cn
(J. Chang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.020

Q. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 4178e4183
4179
a novel potent NRTI that could overcome drug-resistance problems
[19e21].
2. Chemistry
The synthesis of 10 is illustrated in Scheme 1. According to the
reported method [8,13,19e25], treatment of commercially available
1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-
HBr-HOAc (45%) in dichloromethane (DCM) gave exclusively the
bromide 2, which was coupled with silylated uracil in chloroform to
provide the -nucleoside analog 3 in a good yield after recrystal-
D
-arabinofuranoside (1) with
a-
Fig. 1. Structures of 1-(40-Azido-20-deoxy-20-fluoro-
b-D-arabinofuranosyl)cytosine (10)
b
and Its Design Lead 40-Azido-thymidine.
lization. Deprotection of 3 with methanolic ammonia afforded
nucleoside 4 in excellent yield. The 40-azido substitution was
introduced using the following procedure modified from the
previously reported method [26]. Treatment of 4 with I2/Ph3P in
tetrahydrofuran (THF) followed by elimination in the presence of
sodium methoxide (NaOMe) gave 40-methylene-nucleoside 6.
Treatment of 6 with ICl/NaN3 in THF afforded 40-azido-nucleoside 7.
Benzoylation of 7 followed by treatment with m-chloroperbenzoic
acid (m-CPBA) in the presence of m-chlorobenzoic acid (m-CBA)
yielded a protected 40-azido-uridine analog 9. Treatment of 9 with
triazole/POCl3 followed by methanolic ammonia gave our target 10.
Compound 10 was dissolved in methanol and treated with a solu-
tion of hydrogen chloride in ethyl acetate. The product separated as
a solid and was collected by filtration to afford the hydrochloride
salt (11) of 10. The solid-state structure of 11 was determined by
single-crystal X-ray crystallography (Fig. 2). Crystallographic data
(excluding structure factors) for the structures in this paper have
been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication (nos. CCDC 740473). Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK, (fax: þ44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
HIV mutants that have acquired the ability to discriminate between
ddN and physiologic 20-deoxynucleoside (dN), do not accept ddN
into the active center of their reverse transcriptase (RT), and
selectively cut off the incorporated ddN from their proviral DNA
terminus. Therefore, nucleoside drugs that could prevent the
emergence of drug-resistant HIV variants must have a 30eOH as the
chain terminator of proviral DNA biosynthesis.
A 40-C-substituted-20-deoxynucleoside (40sdN) satisfies these
conditions, because 40sdN has all of the functional groups of dN,
making it difficult for HIV to discriminate between a 40sdN and dN.
At the same time, the 30eOH group of 40sdN would not be useful for
elongation of proviral DNA biosynthesis. Thus, 40sdN could be the
chain terminator of proviral DNA biosynthesis and active against
HIV as well as HIV strains resistant to current ddN drugs. In addi-
tion, modification of the carbohydrate moiety of nucleosides with
electron-withdrawing groups can greatly affect the nucleoside’s
electronic properties and conformational shape [8e14], which
often leads to dramatically improved activities. Therefore, we
incorporated a fluorine atom, which has the strongest electron-
withdrawing property of the halogens atoms, into our newly
designed 40sdN. Moreover, azido, cyano, and ethynyl groups, which
have characteristic electronic and structural features due to their sp
hybridization, are present in many biologically active compounds
[15e17]. It has been proposed that the furanose ring of 40-C-
azidothymidine exhibits an unnatural 30-C-endo conformation, and
the compound was active against multidrug-resistant strains of HIV
3. Results and discussion
The anti-HIV activity of 10 and 11 was first evaluated in vitro
according to standard procedures using efavirenz (EFV) and zido-
vudine (AZT) as controls (Table 1) [27e30]. Compounds 10 and 11
exhibited extremely potent antiviral activity with EC50 values of 0.3
and 0.13 nM, respectively, compared with EFV (EC50: 1.2 nM) and
AZT (EC50: 47 nM). The increased activity of 11 compared with 10
likely reflects its increased solubility of 11 in water [31]. Both
(EC50: 0.34 mM) [18]. Therefore, we were interested in designing
novel 40sdN compounds with such substitutions at the 40-position.
Herein, we report the synthesis and evaluation of 1-(40-azido-20-
deoxy-20-fluoro-
b-D-arabinofuranosyl)cytosine (10) (Fig. 1) as
Scheme 1. Reagents and conditions: a) HBr/HOAc/DCM; b) silylated uracil(5-fluorouracil)/CHCl3; c) NH3/MeOH; d) Ph3P/I2/DMF; e) NaOMe/THF; f) ICl/NaN3/THF; g) BzCl/Pyr.DCM;
h) m-CPBA/m-CBA/DCM; i) 1. triazole/POCl3/pyridine/DCM, 2. NH4OH, 3. NH3/MeOH; j) HCl, MeOH, EtOAc.

4180
Q. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 4178e4183
Table 2
Anti-HIV activity of 11 against NL4-3 (wild-type), NL4-3
(K101E), and RTMDR.
Compound 11
EC50 (nM)a
NL4-3
NL4-3.K101E
HIV-1RTMDR
0.086
0.15
0.11
a
EC50 (nM) is the concentration that inhibits HIV by
50%. Results were averaged from at least two separate
experiments.
(Table 4). A Promega CellTiter-Glo Luminescent Cell Viability Assay
was used to determine the number of viable cells based on quan-
titation of the presence of ATP, which signals the presence of
metabolically active cells. Compound 11 showed little cytotoxicity
against TZM-BL, HEPG-2, and 293T cells with IC50 values over
140
Comparatively, 11 showed some cytotoxicity against MT4, ACH-2,
and PBMC cells with IC50 values of 0.12, 0.11, and 0.15 M, and cor-
mM, resulting in therapeutic index (TI) values of >280,000.
m
responding TI values of 1418,1255, and 1709. Overall,11 showed less
toxicity to adherent cells compared to suspension cells. Neverthe-
less, the TI values were still greater than at least one thousand.
Regarding mechanism of action, compound 10 is likely to have
a strong binding in the active site of HIV RT due to the introduction
of fluorine at the 20-position. Although compound 10 possesses
a 30-hydroxy group, it might act like a DNA chain terminator similar
to 40-azido thymidine (Fig. 3). Based on the drug profile shown in
Table 3 against resistant virus strains, D67 and M184 are likely the
two critical amino acid residues in the NRTI binding site of HIV-1 RT
that interact intimately with the compound. NRTI-resistant viruses
with D67N or M184V mutation are markedly resistant to
compound 10. Thus, the compound is likely to bind between the
palm and the finger subdomains of HIV-1 RT and make critical
interactions with residues including D67 and M184.
Fig. 2. X-ray crystallographic structure of hydrochloride salt 11.
compounds did not show significant cytotoxicity in concentrations
up to 100 M against the MT-2 cell line using the MTT assay. In the
m
further evaluation of 11, we discovered that it retained its sub-
nanomolar activity against drug-resistant HIV strains NL4-3
(K101E) and RTMDR (Table 2). K101E tends to decrease viral
susceptibility to all nucleoside RT inhibitors, while RTMDR is
a multiple RT inhibitor-resistant strain, which is insensitive to AZT,
ddI, nevirapine, and other NNRTIs. In our screening, 11 exhibited
extremely potent anti-HIV activity against NL4-3 (wild-type), NL4-
3 (K101E), and RTMDR, with EC50 values of 0.086, 0.15, and 0.11 nM,
respectively. These findings suggested that 11 has a great potential
to be developed as a novel NRTI.
4. Conclusion
Therefore, in the following study, we further evaluated the anti-
HIV potential of 11 using a panel of eleven NRTI-resistant viral
strains from the NIH Reagent Program. As shown in Table 3, 11
retained its nanomolar activity against resistant strains 7324-1,
7324-4, 7303-3, 35764-2, and 56252-1 with EC50 values of 0.595,
0.735, 0.56, 0.42, 0.525 nM, respectively, which were similar to that
of 11 against the wild-type NL4-3 (EC50: 0.318 nM). However, 11
partially or completely lost its nanomolar inhibitory activity against
10076-4, 7295-1, 4755-5, 6463-13, 1617-1, and 29129-2. After
carefully analyzing the mutations of these resistant viral strains, we
discovered that one single mutation M184V may lead to the
substantially reduced antiviral activity of 11. This M184V mutation
in RT was discovered previously to be 3 TC treatment-associated,
and it was also found that the M184V mutation increases the HIV
sensitivity to AZT in AZT-resistant viruses, suggesting that
compound 11 may be used in combination with AZT to reduce the
influence of resistance issues.
In summary, novel 40sdN compounds 10 and 11 were designed,
synthesized, and evaluated for anti-HIV activity. The hydrochloride
salt 11 retained its sub-nanomolar activity against NRTI-resistant
(K101E) and multi-drug-resistant HIV strains (RTMDR). In further
studies, 11 also showed nanomolar activity against resistant viral
strains, except for the M184V single mutation. Compound 11
showed little cytotoxicity against adherent cells and marginal
cytotoxicity against suspension cells with TI values over one
thousand. Overall, compound 11 merits further development as an
anti-AIDS clinical trial candidate.
Table 3
Anti-HIV activity of compound 11 against NIH reagent program NRTI-resistant virus
panel.
NRTI-resistant virus panel
EC50 (nM)a
7324e1 (M41L, D67N, K70R, T215F, K219E, T69N)
7324e4 (M41L, K70R, T215F, K219E)
10076e4 (M41L, T215Y, M184V)
0.595
The cytotoxicity profiles of 11 were further evaluated using six
human cell lines different from the previously used MT-2 cell line
0.735
>40,000b
>40,000
7295e1 (D67N, K70R, T215F, K219Q, M184V, T69N)
4755e5 (M41L, D67N, L210W, T215Y, M184V, T69D, E44D, V118I) >40,000
Table 1
6463e13 (M41L, D67N, L210W, T215Y, M184V, V118I)
7303e3 (M41L, D67N, L210W, T215Y, T69D, E44D, V118I)
1617e1 (K70G, M184V, T69K, V75I, F77L, F116Y, Q151M)
35764e2 (V75I, F77L, F116Y, Q151M)
>40,000
0.56
32.2
Anti-HIV activity of 10 and 11 against wild-type HIV-1 virus.
Compound
EC50 (nM) (wild-type)a
0.42
10
11
EFV
AZT
0.3
0.13
1.2
29129e2 (M41L, D67N, L210W, T215Y, M184V)
56252e1 (K70R, V75I, F77L, F116Y, Q151M, K65R)
NL4-3 (wild-type)
>40,000
0.525
0.318
47
a
EC50 (nM) is the concentration that inhibits HIV by 50%. Results were averaged
from at least two separate experiments.
a
EC50 (nM) is the concentration that inhibits HIV by 50%. Results
were averaged from at least two separate experiments.
b
The highest concentration tested was 40,000 nM.

Q. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 4178e4183
4181
Table 4
A mixture of uracil (12.6 g, 113.0 mmol) and (NH4)2SO4 (1.57 g)
in hexamethyldisilazane (HMDS) (1.5 L) was refluxed under
nitrogen for 20 h, and the homogeneous solution obtained was
evaporated to dryness under vacuum to give the silylated uracil. To
the silylated uracil residue was added a solution of compound 2 in
CHCl3 (800 mL) and the mixture was refluxed under nitrogen for
24 h. The reaction was quenched by addition of ice-water and
filtered. The aqueous layer was extracted with DCM (3 ꢁ 200 mL)
and the combined organic solution was washed with brine, dried
over Na2SO4, filtered, and concentrated to give a white solid, which
was recrystallized with 5% EtOH-DCM to give compound 3 (16.7 g,
Cytotoxicity screening of compound 11 against six human cell lines.
Cells
IC50 (m
M)a
TZM-BL (derived from Hela Cells)
HEPG-2
293T
MT4
ACH-2
PBMC (PHA activated)
>140b
>140
>140
0.12
0.11
0.15
a
IC50 (mM) is the concentration that inhibits cell growth by 50%. Results
were averaged from at least three separate experiments.
b
The highest concentration tested was 140 mM.
84.2%). 1H NMR (300 MHz, CDCl3)
d 8.35 (1H, brs, NH), 7.43e8.11
(11H, m, AreH, H-6), 6.33 (1H, dd, J ¼ 21.59, 2.93 Hz, H-10), 5.69 (1H,
d, J ¼ 8.05 Hz, H-5), 5.63 (1H, dd, J ¼ 17.20, 2.93 Hz, H-30), 5.34 (1H,
dd, J ¼ 50.13, 2.93 Hz, H-20), 4.78 (2H, d, J ¼ 4.39 Hz, H-50), 4.52 (1H,
m, H-40); ESI-MS: m/z 455 [M þ H]þ; Anal. calcd (C23H19FN2O7): C,
60.79; H, 4.21; N, 6.16; found: C, 60.75; H, 4.24; N, 6.11.
5. Experimental
All chemical reagents were commercially available and column
chromatography was performed on silica gel 200e300 mesh
(Yantai Silica Gel Co. LTD). Analytical TLC was performed on silica
gel GF254. Melting points were recorded with an XT-4 melting point
apparatus and are uncorrected. IR spectra were obtained on
a NICOLET 6700 spectrometer. 1H, 19F, and 13C NMR spectra were
obtained with a Bruker AV-300 spectrometer. Chemical shifts are
5.2. Synthesis of 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil (4)
A solution of compound 3 (7.79 g, 17.1 mmol) in saturated NH3-
MeOH (200 mL) was stirred at room temperature for 24 h and
evaporated to dryness under reduced pressure. DCM (25 mL) was
added to the residue and the mixture was stirred at room
temperature for 1 h. The white solid was collected by filtration to
give compound 4 (3.93 g, 93.0%). 1H NMR (300 MHz, DMSO-d6)
reported as d (ppm) downfield with respect to an internal standard
of tetramethylsilane (TMS). High-resolution mass spectra (HRMS-
ESI) were obtained on a MicroÔ Q-TOF Mass Spectrometer.
Elemental analyses were performed with a Carlo-Erba 1106 C, H,
and N analyzer. LC-MS spectra were measured on an Agilent MSD-
1100 ESI-MS/MS system. X-ray crystallography was obtained on
Rigaku Saturn 724 CCD diffractometer.
d
11.35 (1H, brs, NH), 7.71 (1H, dd, J ¼ 8.05, 1.46 Hz, H-6), 6.10 (1H,
dd, J ¼ 16.10, 4.39 Hz, H-10), 5.87 (1H, d, J ¼ 5.12 Hz, 30eOH), 5.64
(1H, d, J ¼ 8.05 Hz, H-5), 5.08 (1H, t, J ¼ 5.85 Hz, 50eOH), 5.03 (1H,
ddd, J ¼ 52.69, 4.03, 2.93 Hz, H-20), 4.21 (1H, dm, J ¼ 19.76 Hz, H-30),
3.79 (1H, m, H-40), 3.60 (2H, m, H-50); ESI-MS: m/z 269 [M þ Na]þ;
Anal. calcd (C9H11FN2O5): C, 43.91; H, 4.50; N, 11.38; found: C,
43.90; H, 4.29; N, 11.33.
5.1. Synthesis of 1-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-
arabino-furanosyl)uracil (3)
b-D-
To a solution of 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-D-arabi-
nofuranoside (1) (20.0 g, 43.1 mmol) in anhydrous DCM (400 mL)
was added HBreHOAc (45% v/v, 50 mL, 271 mmol) dropwise under
nitrogen at 0 ꢀC and the resulting solution was stirred at room
temperature for 20 h. The solution was washed with saturated
NaHCO3 (3 ꢁ 100 mL), dried over Na2SO4, filtered, and concentrated
to give compound 2 as syrup, which was used in the next step
without further purification.
5.3. Synthesis of 1-(2,5-dideoxy-2-fluoro-5-iodo-
furanosyl)uracil (5)
b-D-arabino-
To a solution of compound 4 (3.73 g, 15.2 mmol), imidazole
(2.06 g, 30.3 mmol), and triphenylphosphine (5.96 g, 22.7 mmol) in
THF (100 mL) was added a solution of iodine (5.77 g, 22.7 mmol) in
Fig. 3. Mechanism of phosphorylated compound 10.

4182
Q. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 4178e4183
THF (50 mL) dropwise at 0 ꢀC. The reaction mixture was stirred at
room temperature overnight and quenched with a saturated solu-
tion of Na2SO3, extracted with EtOAc (2 ꢁ 100 mL). The combined
organic solution was washed with water, brine, dried over Na2SO4,
filtered, and concentrated to yield a thick oil, which was purified by
silica gel column chromatography (1e5% MeOH in DCM) to afford
concentrated. The residue was purified by silica gel column chro-
matography (0.1e1% MeOH in DCM) to afford compound 8 (1.2 g,
81.2%). IR (KBr): 2118 cmꢂ1(N3); 1H NMR (300 MHz, DMSO-d6)
d
11.63 (1H, br s, NH), 7.57e8.09 (6H, m, AreH and H-6), 6.52 (1H,
dd, J ¼ 18.69, 5.52 Hz, H-10), 6.07 (1H, dd, J ¼ 21.57, 4.05 Hz, H-30),
5.82 (1H, ddd, J ¼ 52.21, 5.32, 4.38 Hz, H-20), 5.78 (1H, d, J ¼ 8.14 Hz,
H-5), 3.99 (2H, q, J ¼ 11.66 Hz, H-50); ESI-MS: m/z 524 [Mþ23]þ;
Anal. calcd (C16H13FIN5O5): C, 38.34; H, 2.61; N, 13.97; found: C,
38.26; H, 2.48; N, 13.85.
compound 5 (4.51 g, 83.5%). 1H NMR (300 MHz, DMSO-d6)
d 11.50
(1H, br s, NH), 7.58 (1H, dd, J ¼ 8.14, 1.95 Hz, H-6), 6.17 (1H, dd,
J ¼ 18.40, 3.99 Hz, H-10), 6.15 (1H, br, 30eOH), 5.67 (1H, d, J ¼ 8.14Hz,
H-5), 5.08 (1H, ddd, J ¼ 52.41, 3.84, 2.55 Hz, H-20), 4.14 (1H, ddd,
J ¼ 19.68, 4.21, 2.65 Hz, H-30), 3.87 (1H, dd, J ¼ 11.02, 5.19 Hz, H-40),
5.7. Synthesis of 1-(4-azido-2-deoxy-2-fluoro-3-O-benzoyl-5-O-(3-
chlorobenzoyl)-b-D-arabinofuranosyl)uracil (9)
3.43e3.57 (m, 2H); 13C NMR (75 MHz, DMSO-d6)
d
6.2, 76.7, 82.0,
83.0, 95.3, 101.4, 141.2, 150.0, 162.8; ESI-MS: m/z 379 [Mþ23]þ;
Anal. calcd (C9H10FIN2O4): C, 30.36; H, 2.83; N, 7.87; found: C,
30.30; H, 2.88; N, 7.84.
To a solution of 8 (1.10 g, 2.19 mmol) in DCM (100 mL) and water
(60 mL) were added successively K2HPO4 (0.76 g, 4.38 mmol), n-
Bu4NHSO4 (0.82 g, 2.41 mmol), and m-CBA (0.38 g, 2.41 mmol). The
reaction mixture was cooled to 0 ꢀC and m-CPBA (77%, 1.47 g,
6.57 mmol) was added. The reaction mixture was stirred at room
temperature overnight. Na2SO3 was added and the mixture was
extracted with EtOAc (500 mL). The combined organic solution was
washed with Na2SO3 (5%), brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified by silica gel column chro-
matography (0.1e0.5% MeOH in DCM) to provide compound 9
(667 mg, 57.5%). IR (KBr): 2117 cmꢂ1 (N3); 1H NMR (300 MHz,
5.4. Synthesis of 1-(2,5-dideoxy-2-fluoro-b-D-arabino-4-eno-
furanosyl)uracil (6)
A solution of compound 5 (4.2 g, 11.8 mmol) in dry MeOH (40 mL)
containing MeONa (25%wt in MeOH, 10.2 mL, 47.3 mmol) was heated
to 65 ꢀC for 3 h and an additional aliquot of MeONa (25%wt in MeOH,
3.4 mL) was added. After 30 min, the reaction mixture was allowed to
stay at room temperature overnight and brine was added. The
mixture was adjusted to pH 3 by addition of 1N HCl and the mixture
was extracted with EtOAc (2 ꢁ 300 mL). The combined organic
solution was dried over Na2SO4, filtered, and concentrated. The
residue was purified by silica gel column chromatography (2e5%
MeOH in DCM) to afford compound 6 (1.94 g, 72.1%). 1H NMR
CDCl3)
d
7.41e8.10 (10H, m, AreH and H-6), 6.63(1H, dd, J ¼ 18.36,
3.61 Hz, H-10), 5.73e5.84(2H, m, H-30 and H-5), 5.45 (1H, ddd,
J ¼ 51.37, 3.55, 2.03 Hz, H-20), 4.86 (2H, q, J ¼ 11.87 Hz, H-50); ESI-
MS: m/z 553 [Mþ23]þ; Anal. calcd (C23H17FClN5O7): C, 52.14; H,
3.23; N, 13.22; found: C, 52.03; H, 3.22; N, 13.00.
(300 MHz, CDCl3)
d
9.05(1H, br s, NH), 7.32(1H, dd, J ¼ 8.16, 2.31 Hz, H-
6), 6.62 (1H, dd, J ¼ 20.97, 2.79 Hz, H-10), 5.76 (1H, d, J ¼ 8.22Hz, H-5),
5.09 (1H, dd, J ¼ 51.44, 2.95 Hz, H-20), 4.76 (1H, d, J ¼ 2.74 Hz, ¼ CH2),
4.73 (1H, d, J ¼ 11.46 Hz, H-30), 4.52 (1H, d, J ¼ 2.74Hz, ¼ CH2); ESI-MS:
m/z 227 [M ꢂ H]þ; Anal. calcd (C9H9FN2O4): C, 47.37; H, 3.98; N,12.28;
found: C, 47.43; H, 3.86; N, 12.13.
5.8. Synthesis of 1-(4-azido-2-deoxy-2-fluoro)-
b-D-
arabinofuranosyl)cytosine (10)
To a solution of compound 9 (0.40 g, 0.75 mmol) in DCM
(15 mL) was added 1,2,4-triazole (0.57 g, 8.25 mmol) followed by
pyridine (0.67 mL, 8.25 mmol). The solution was cooled to 0 ꢀC and
POC13 (0.39 mL, 3.75 mmol) was added. The solution was stirred at
5.5. Synthesis of 1-(4-azido-2,5-dideoxy-2-fluoro-5-iodo-
arabino-furanosyl)uracil (7)
b-D-
0
ꢀC for 2 h then room temperature overnight. The reaction was
quenched with water and the mixture was extracted with DCM
(2 ꢁ 250 mL). The combined organic solution was dried over
Na2SO4, filtered, and concentrated. The residue was dissolved in
THF (40 mL) and NH4OH (33%, 40 mL) was added. The reaction
mixture was stirred at room temperature for 1 h. Solvent was
removed and the residue was dissolved in MeOH (40 mL) and 7N
NH3 in MeOH (40 mL) was added. The solution was stirred at room
temperature overnight. The solution was concentrated to dryness
under reduced pressure and the residue was purified by silica gel
column chromatography (1e5% MeOH in DCM) to give compound
10 (92 mg, 41.1%) as a white solid. mp: 86e87 ꢀC; IR (KBr):
To a suspension of NaN3 (1.38 g, 21.3 mmol) in THF (50 mL) at
ꢀC was added ICl (2.31 g, 14.2 mmol). After 10 min, a solution of
0
compound 6 (1.62 g, 7.10 mmol) in THF (16 mL) was added drop-
wise to the mixture and the reaction mixture was stirred at room
temperature overnight. Aqueous Na2SO3 solution was added and
the mixture extracted with EtOAc (3 ꢁ 250 mL). The combined
organic solution was dried over Na2SO4, filtered, and concentrated.
The residue was purified by silica gel column chromatography
(1e5% MeOH in DCM) to afford compound 7 (1.82 g, 64.6%). IR KBr):
2119 cmꢂ1(N3); 1H NMR (300 MHz, MeOH-d4)
d 7.68 (1H, dd,
J ¼ 8.16, 1.87 Hz, H-6), 6.46 (1H, dd, J ¼ 16.07, 4.39 Hz, H-10),
5.74(1H, d, J ¼ 8.12 Hz, H-5), 5.22 (1H, ddd, J ¼ 52.93, 4.36, 3.30 Hz,
H-20), 4.65 (1H, dd, J ¼ 18.67, 3.31 Hz, H-30), 3.69 (2H, m, H-50); ESI-
MS: m/z 420 [Mþ23]þ; Anal. calcd (C9H9FIN5O4): C, 27.22; H, 2.28;
N, 17.64; found: C, 27.09; H, 2.23; N, 17.73.
2118 cmꢂ1 (N3); 1H NMR (300 MHz, MeOH-d4)
d 7.80 (1H, d,
J ¼ 7.57Hz, H-6), 6.50 (1H, dd, J ¼ 12.10, 4.67 Hz, H-10), 5.97 (1H, d,
J ¼ 7.57Hz, H-5), 5.21 (1H, dt, J ¼ 53.52, 4.48 Hz, H-20), 4.49 (1H, dd,
J ¼ 21.63, 4.34 Hz, H-30), 3.84 (2H, s, H-50); 13C NMR (75.47 MHz,
MeOH-d4)
d
63.4, 76.5 (J ¼ 25.2 Hz), 84.8 (J ¼ 17.2 Hz), 96.1, 96.2
(J ¼ 193.4 Hz), 98.6 (J ¼ 7.5 Hz), 143.0, 157.9, 167.8; 19F NMR
(282.4 MHz, MeOH-d4): ꢂ202.5; ESI-TOF/MS for C9H11FN6O4: calcd
287.0904 [M þ H]þ; found: 287.0905[M þ H]þ; Anal. calcd
(C9H11FN6O4): C, 37.77; H, 3.87; N, 29.36; found: C, 37.53; H, 3.90;
N, 29.19.
5.6. Synthesis of 1-(4-azido-2,5-dideoxy-2-fluoro-3-O-benzoyl-5-
iodo-b-D-arabinofuranosyl)uracil (8)
To a solution of compound 7 (1.17 g, 2.95 mmol) in DCM
(130 mL) were added Et3N (0.82 mL, 5.90 mmol) and a catalytic
amount of 4-dimethylaminopyridine. The reaction mixture was
cooled to 0 ꢀC and benzyl chloride (0.41 mL, 3.54 mmol) was added.
After 20 min, water and 2M K2CO3 were added, and the mixture
was extracted with EtOAc (400 mL). The organic layer was washed
5.9. Synthesis of 1-(4-azido-2-deoxy-2-fluoro)-
b-D-
arabinofuranosyl)cytosine hydrochloride salt (11)
Compound 10 (50 mg, 0.17 mmol) was dissolved in MeOH (1 mL)
with brine (2
ꢁ
50 mL), dried over Na2SO4, filtered, and
and treated with a solution of hydrogen chloride (20 mmol) in

Q. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 4178e4183
4183
EtOAc (10 mL). The product separated as a solid and was collected
by filtration to afford the hydrochloride salt of compound 10
(34 mg, 61.1%). IR (KBr): 2117 cmꢂ1 (N3); 1H NMR (D2O)
7.94 (1H,
Acknowledgements
d
J. Chang thanks the National Natural Science Foundation of
China (#20672030; #30825043) for financial support. Thanks are
also due to a grant AI-33066 from the National Institute of Allergy
and Infectious Diseases (NIAID) awarded to K.H. Lee.
dd, J ¼ 8.05, 1.10 Hz, H-6), 6.54 (1H, dd, J ¼ 10.98, 5.12 Hz, H-10), 6.26
(1H, d, J ¼ 8.05 Hz, H-5), 5.39 (1H, dt, J ¼ 52.69, 5.12 Hz, H-20), 4.58
(1H, dd, J ¼ 21.59, 4.76 Hz, H-30), 3.97 (2H, q, J ¼ 12.66 Hz, H-50); ESI-
MS: m/z 287 [M þ H]þ.
References
5.10. Single-crystal X-ray diffraction analysis of hydrochloride salt 11
C9H11FN6O4$HCl, fw ¼ 322.70, Monoclinic P2(1), a ¼ 6.5738(13)
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a
¼
g
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compound 11 is 14.3 g in 100 g water.
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