Synthesis and Cytostatic and Antiviral Activities of 2′-Deoxy-2′,2′-difluororibo- and 2′-Deoxy-2′-fluororibonucleosides Derived from 7-(Het)aryl-7-deazaadenines

Article abstract of DOI:10.1002/cmdc.201300047

A series of sugar-modified derivatives of cytostatic 7-heteroaryl-7-deazaadenosines (2′-deoxy-2′-fluororibo- and 2′-deoxy-2′,2′-difluororibonucleosides) bearing an aryl or heteroaryl group at position7 was prepared and screened for biological activity. The difluororibonucleosides were prepared by non- stereoselective glycosidation of 6-chloro-7-deazapurine with benzoyl-protected 2-deoxy-2,2-difluoro-D-erythro-pentofuranosyl-1-mesylate, followed by amination and aqueous Suzuki cross-couplings with (het)arylboronic acids. The fluororibo derivatives were prepared by aqueous palladium-catalyzed cross-coupling reactions of the corresponding 7-iodo-7-deazaadenine 2′-deoxy-2′-fluororibonucleoside 20 with (het)arylboronic acids. The key intermediate 20 was prepared by a six-step sequence from the corresponding arabinonucleoside by selective protection of 3′- and 5′-hydroxy groups with acid-labile groups, followed by stereoselective S_N2 fluorination and deprotection. Some of the title nucleosides and 7-iodo-7-deazaadenine intermediates showed micromolar cytostatic or anti-HCV activity. The most active were 7-iodo and 7-ethynyl derivatives. The corresponding 2′-deoxy-2′,2′-difluororibonucleoside 5′-O-triphosphates were found to be good substrates for bacterial DNA polymerases, but are inhibitors of human polymeraseα. Sugar modified: Fluorinated analogues of 7-(het)aryl-7-deazaadenines were synthesized, and these nucleosides showed micromolar cytostatic activity against cancer cell lines and moderate anti-HCV activity. The corresponding nucleoside 5′-O-triphosphates were found to be good substrates for bacterial DNA polymerases, but are inhibitors of human polymeraseα.

Full text of DOI:10.1002/cmdc.201300047

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DOI: 10.1002/cmdc.201300047
Synthesis and Cytostatic and Antiviral Activities of 2’-
Deoxy-2’,2’-difluororibo- and 2’-Deoxy-2’-
fluororibonucleosides Derived from 7-(Het)aryl-7-
deazaadenines
Pavla Perlꢀkovꢁ,[a] Ludovic Eberlin,[a] Petra Mꢂnovꢁ,[a] Veronika Raindlovꢁ,[a]
[a]
[a]
[b]
[b]
[a, c]
ˇ
ˇ
ˇ
Lenka Slavetꢀnskꢁ, Eva Tloustovꢁ, Gina Bahador, Yu-Jen Lee, and Michal Hocek*
A series of sugar-modified derivatives of cytostatic 7-hetero-
aryl-7-deazaadenosines (2’-deoxy-2’-fluororibo- and 2’-deoxy-
2’,2’-difluororibonucleosides) bearing an aryl or heteroaryl
group at position 7 was prepared and screened for biological
activity. The difluororibonucleosides were prepared by non-
stereoselective glycosidation of 6-chloro-7-deazapurine with
benzoyl-protected 2-deoxy-2,2-difluoro-d-erythro-pentofurano-
syl-1-mesylate, followed by amination and aqueous Suzuki
cross-couplings with (het)arylboronic acids. The fluororibo de-
rivatives were prepared by aqueous palladium-catalyzed cross-
coupling reactions of the corresponding 7-iodo-7-deaza-
adenine 2’-deoxy-2’-fluororibonucleoside 20 with (het)arylbor-
onic acids. The key intermediate 20 was prepared by a six-step
sequence from the corresponding arabinonucleoside by selec-
tive protection of 3’- and 5’-hydroxy groups with acid-labile
groups, followed by stereoselective SN2 fluorination and de-
protection. Some of the title nucleosides and 7-iodo-7-deaza-
adenine intermediates showed micromolar cytostatic or anti-
HCV activity. The most active were 7-iodo and 7-ethynyl deriva-
tives. The corresponding 2’-deoxy-2’,2’-difluororibonucleoside
5’-O-triphosphates were found to be good substrates for bac-
terial DNA polymerases, but are inhibitors of human polymer-
ase a.
Introduction
Within the framework of our long-term project on the synthe-
sis and biological activity screening of modified purine and 7-
deazapurine ribonucleosides,[1] we recently discovered a new
group[2] of nucleoside cytostatics, 7-heteroaryl-7-deazaadenine
ribonucleosides 2 (Figure 1), as derivatives of natural cytotoxic
antibiotic tubercidin (1). Nucleosides 2 exerted a significant in
vitro cytostatic effect against a broad spectrum of leukemia
and tumor cell lines at nanomolar concentrations, as well as
nonselective anti-HCV activities (probably caused by cytotoxici-
ty). More recently,[3] we prepared their 2’-C-methylribonucleo-
side, arabinonucleoside, and 2’-fluoroarabinonucleoside deriva-
tives 3 (Figure 1). These sugar-modified derivatives showed
low micromolar cytostatic or anti-HCV activities, indicating that
the ribo configuration of the sugar is not crucial for activity. To
complement the structure–activity relationship of this class of
biologically active nucleosides, herein we report sugar-modi-
fied derivatives containing a fluorine atom in the ribo configu-
ration. The 2’,2’-difluororibose motif is inspired by cytostatic
gemcitabine[4] (2’,2’-difluorodeoxycytidine, 4) which is a drug in
ˇ
[a] Dr. P. Perlꢀkovꢁ, L. Eberlin, P. Mꢂnovꢁ, V. Raindlovꢁ, Dr. L. Slavetꢀnskꢁ,
ˇ
ˇ
E. Tloustovꢁ, Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Gilead Sciences & IOCB Research Center
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
E-mail: hocek@uochb.cas.cz
[b] Dr. G. Bahador, Dr. Y.-J. Lee
Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404 (USA)
[c] Prof. Dr. M. Hocek
Department of Organic Chemistry, Faculty of Science
Charles University in Prague, Hlavova 8, 12843 Prague 2 (Czech Republic)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300047.
Figure 1. Biological activity of 7-deazaadenine nucleosides and gemcitabine.
ChemMedChem 2013, 8, 832 – 846 832
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clinical use for the treatment of solid tumors (pancreas and
lung cancer). Some 2’-deoxy-2’-fluororibonucleosides are also
known cytostatics.[5]
Unprotected nucleoside 9 was used as starting material for
a series of palladium-catalyzed Suzuki cross-coupling reactions
with various (het)arylboronic acids under aqueous-phase con-
ditions[8] to synthesize 7-(het)aryl-7-deazaadenine 2’-deoxy-
2’,2’-difluoro-b-d-erythro-pentofuranosyl nucleosides 11 a–g in
moderate to good yields (39–71%; Scheme 2). The cross-cou-
plings were performed in the presence of Na2CO3, triphenyl-
phosphine-3,3’,3’’-trisulfonic acid trisodium salt (TPPTS),
Pd(OAc)2 in water/acetonitrile 2:1 at 1008C.
Results and Discussion
Synthesis
The synthetic approach to the target 7-heteroaryl-7-deazaade-
nine 2’-deoxy-2’,2’-difluoro-d-erythro-pentofuranosyl nucleo-
sides relied on the glycosidation of 6-chloro-7-iodo-7-deaza-
purine (6) with commercially available difluorosugar mesylate
5. However, the TMSOTf-catalyzed glycosylation of 6 with 5 ac-
cording to a published procedure[6] failed. Therefore, we fo-
cused on nucleobase anion glycosylation, which was previous-
ly used for the synthesis of 7-deazapurine 2’-deoxy-2’,2’-difluo-
roribonucleosides.[7] The reaction of 6 with mesylate 5 was per-
formed in the presence of tris[2-(2-methoxyethoxy)ethyl]amine
(TDA-1; 1 equiv) and potassium hydroxide (2 equiv) in aceto-
nitrile at 508C (Scheme 1). Under these conditions, the reaction
Scheme 2. Reagents and conditions: a) R-B(OH)2, Na2CO3, TPPTS, Pd(OAc)2,
H2O/CH3CN (2:1), 2 h, 1008C.
The (trimethylsilyl)ethynyl derivative 12 was prepared in
75% yield by the Sonogashira reaction of nucleoside 9 with
(trimethylsilyl)acetylene in the presence of copper iodide, trie-
thylamine, and PdCl2(PPh3)2 in N,N-dimethylformamide (DMF).
Subsequent desilylation using potassium carbonate in metha-
nol provided ethynyl derivative 11 h in 60% yield (Scheme 3).
Scheme 1. Reagents and conditions: a) TDA-1, KOH, CH3CN, 4 days, 508C;
b) NH3(aq) (25% w/w), dioxane, 20 h, 1208C.
proceeded with incomplete conversion to form both anomers
of 2’-deoxy-2’,2’-difluoro-d-erythro-pentofuranosyl nucleosides
7 and 8 in low yields (18% total, b/a ratio ~1:1). At higher
temperature (808C) the overall yield of nucleosides 7 and 8
was improved to 27%, but the b/a ratio increased to 1:3.6. On
the other hand, a greater excess of KOH (3.5 equiv) led to the
formation of anomers 7 and 8 in 2:1 b/a ratio, but the overall
yield dropped to 6%. Nucleoside anomers 7 and 8 were sepa-
rable only by two successive column chromatographic steps
with very slow gradient. Therefore, the glycosylation reaction
was performed on a larger scale (10 g), and the pure anomers
7 and 8 were isolated in low yields (8 and 4%, respectively),
but in sufficient quantities for subsequent steps. Benzoylated
anomeric 6-chloro-7-iodo-7-deazapurine nucleosides 7 and 8
were subjected to a simultaneous amination and deprotection
reaction by treating with aqueous ammonia in dioxane in
a steel pressure flask to obtain free nucleosides 9 and 10 in
good yields (62 and 88%, respectively; Scheme 1).
Scheme 3. Reagents and conditions: a) (trimethylsilyl)acetylene, PdCl2(PPh3)2,
CuI, Et3N, DMF, 18 h, RT; b) K2CO3, CH3OH, 1 h, RT.
Analogously, the Suzuki cross-coupling reaction was also
used for the introduction of (het)aryl groups into position 7 of
a-anomeric 7-iodo-7-deazaadenine nucleoside 10. The corre-
sponding 7-(het)aryl-7-deazaadenine 2’-deoxy-2’,2’-difluoro-a-
d-erythro-pentofuranosyl nucleosides 13a–e were prepared in
38–65% yields (Scheme 4).
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90% trifluoroacetic acid (TFA) to give free 2’-deoxy-2’-fluorori-
bonucleoside 20 (36% over two steps). The relative configura-
tion of fluorine in compound 20 was established by analysis of
1
H,H and H,F coupling constants in the H NMR spectra; cou-
pling constants were identical to those observed for 2’-deoxy-
2’-fluororibonucleosides, reported previously.[7]
The 7-iodo-7-deazaadenine fluororiboside 20 was then used
as a starting material for a series of Suzuki cross-coupling reac-
tions with (het)aryl boronic acids under standard conditions. In
this way, the title 7-heteroaryl-7-deazaadenosine 2’-deoxy-2’-
fluororibonucleosides 21a–e were synthesized in 53–69%
yields (Scheme 6). The additional ethynyl derivative 21h was
prepared by Sonogashira reaction with (trimethylsilyl)acetylene
(76%). The trimethylsilyl group was spontaneously cleaved
during hydrolytic reaction workup (Scheme 6).
Scheme 4. Reagents and conditions: a) R-B(OH)2, Na2CO3, TPPTS, Pd(OAc)2,
H2O/CH3CN (2:1), 2 h, 1008C.
The synthetic approach to 7-heteroaryl-7-deazaadenine 2’-
deoxy-2’-fluororibonucleosides 21 was based on SN2 fluorina-
tion of the corresponding tetrahydropyran-2-yl (THP)-protected
arabinonucleoside (Scheme 5). At first, the known[3] silylated 7-
iodo-7-deazaadenine arabinonucleoside 14 was acetylated in
quantitative yield. Subsequent desilylation of acetate 15 using
Et3N·3HF provided 3’,5’-diol 16 (82%). THP groups were then
introduced at positions 3’ and 5’ to give the bis-THP-protected
acetate 17 in 81% yield. Zemplꢂn deacetylation gave 3’,5’-THP-
protected arabinonucleoside 18 (84%) which was allowed to
react with (diethylamino)sulfur trifluoride (DAST) in the pres-
ence of pyridine in dichloromethane at 08C. The resulting 2’-
deoxy-2’-fluororibonucleoside 19 was directly deprotected by
Scheme 6. Reagents and conditions: a) R-B(OH)2, Na2CO3, TPPTS, Pd(OAc)2,
H2O/CH3CN (2:1), 2 h, 1008C; b) (trimethylsilyl)acetylene, PdCl2(PPh3)2, CuI,
Et3N/DMF, overnight, RT, then hydrolytic workup.
To test substrate activities for DNA polymerases, the corre-
sponding nucleoside 5’-O-triphosphates 11c-TP, 11g-TP, and
11h-TP were synthesized from nucleosides 11 c,g,h by a stan-
dard phosphorylation method,[9] using phosphoryl chloride in
trimethyl phosphate, followed by nucleophilic substitution
with (NHBu3)2H2P2O7 and finally hydrolysis with an aqueous so-
lution of triethylammonium bicarbonate (TEAB). After ion ex-
change and lyophilization from water, the desired triphos-
phates 11c-TP, 11g-TP, and 11h-TP were obtained as sodium
salts (12–36%, Scheme 7). Analogously, the triphosphate of
gemcitabine, 4-TP, was prepared from commercial gemcitabine
hydrochloride.
Biological activity profiling
Cytostatic activity
All the title nucleosides 9, 10, 11 a–h, 13a–e, 20, and 21a–h
were subjected to biological activity screening. The in vitro cy-
totoxic activity was studied on the following cell cultures:
1) human pro-myelocytic leukemia HL60 cells (ATCC CCL 240),
Scheme 5. Reagents and conditions: a) Ac2O, Et3N, DMAP, CH3CN, RT, over-
night; b) Et3N·3HF, THF, RT, overnight; c) DHP, TsOH, DMF, 08C!RT, over-
night; d) MeONa, CH3OH, RT, overnight; e) DAST, pyridine, CH2Cl2, 08C!RT,
overnight; f) 90% TFA, RT, 2 h.
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inactive, whereas the 7-iodo- and 7-ethynyl-7-deazaadenine
derivatives 20 and 21h showed significant cytostatic effects.
This indicates that the mechanism of action in each series may
be distinct, and most likely differs from that of the parent ribo-
nucleosides.
Antiviral activity
The title modified nucleosides were also tested up to 100 mm
for antiviral activities: HIV, RSV, and HCV genotypes 1A, 1B, and
2A replicons.[11] No antiviral activity against HIV or RSV was ob-
served for any of these derivatives. On the other hand, all of
the title nucleosides showed micromolar inhibition in the HCV
replicon assays, although usually accompanied by cytotoxicity
(Table 2). The most promising were the 7-iodo and 7-ethynyl
derivatives 9, 11 h, and 21h which exerted sub-micromolar
EC50 values and cytotoxicities at concentrations higher by one
to two orders of magnitude. These results suggest that, similar-
ly to the previously reported ribonucleosides,[2] these sugar-
modified deazapurine ribonucleosides interfere with critical
cell-growth processes, and most of the activity toward HCV
viral replication is nonspecific.
Scheme 7. Reagents and conditions: a) 1. POCl3, PO(OMe)3, 2.5 h, 08C,
2. (NHBu3)2H2P2O7, tri(n-butyl)amine, DMF, 2.5 h, ꢀ58C, 3. Workup with TEAB
(2m).
2) human cervical carcinoma HeLa S3 cells (ATCC CCL 2.2),
3) human T lymphoblastoid CCRF-CEM cells (ATCC CCL 119),
and 4) hepatocellular carcinoma HepG2 cells (ATCC HB 8065).
Cell viability was determined after incubation for three days
using a metabolic 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide (XTT)-based method.[10] Table 1
summarizes the cytostatic activities. The most active (sub-mi-
cromolar) were both 7-iodo-7-deazaadenine fluoronucleosides
9 and 20. The 2-furyl and 2-thienyl as well as bulky naphthyl
and benzofuryl derivatives of 2’,2’-difluororibonucleosides
11 a,c,g,f showed micromolar cytostatic effects, whereas the 3-
furyl, 3-thienyl, and phenyl derivatives 11 b,d,e were inactive.
This finding is quite surprising, because in the parent ribonu-
cleoside series the 7-deazaadenosines, bearing bulky substitu-
ents at position 7, were inactive.[2] Unexpectedly, one of the a-
anomeric derivatives (13c) also exerted moderate activity. In
the fluororibo series, the 7-(het)aryl derivatives 21a–e were all
Table 2. Anti-HCV activities of fluoronucleosides 9–13, 20, and 21.
Compd
Replicon 1A
Replicon 1B
Replicon 2A
EC50 [mm] CC50 [mm] EC50 [mm] CC50 [mm] EC50 [mm] CC50 [mm]
9
10
1.10
2.48
6.04
16.51
5.30
6.52
8.97
1.43
1.10
0.44
2.04
4.56
1.52
4.71
22.73
1.30
1.98
4.37
2.00
6.52
33.13
0.26
30.66
23.60
>44
>44
32.71
>44
>44
10.36
16.25
17.10
>44
>44
>44
>44
>44
0.06
0.70
4.93
13.18
2.93
5.74
6.49
1.21
1.08
0.50
1.63
1.98
0.44
0.91
2.64
1.02
1.85
5.75
1.99
5.98
18.83
0.47
4.02
14.15
25.47
20.05
9.84
8.91
18.86
2.55
1.12
4.67
5.62
10.30
4.82
7.38
8.58
0.81
1.62
3.29
13.76
14.45
2.24
8.52
10.75
4.36
10.74
14.17
7.96
13.21
20.73
1.86
9.6
23.76
37.06
>44
26.36
>44
41.83
5.99
11 a
11 b
11 c
11 d
11 e
11 f
11 g
11 h
13a
13b
13c
13d
13e
20
21a
21b
21c
21d
21e
21h
4.02
10.94
40.61
8.78
15.62
Table 1. Cytotoxic activities of fluoronucleosides 9–13, 20, and 21.
>44
>44
38.42
>44
>44
>44
Compd
IC50 [mm]
CCRF-CEM
18.91
40.45
43.14
10.20
33.46
37.44
18.53
27.55
HL60
0.42
5.46
7.44
>10
6.33
>10
>10
6.12
3.69
1.41
>10
>10
5.05
HeLa S3
HepG2
9
10
0.64
6.94
9.77
0.42
7.38
6.63
0.35
3.76
5.98
7.37
11 a
11 b
11 c
11 d
11 e
11 f
11 g
11 h
13a
13b
13c
13d
13e
20
>10
>10
>10
>10
>10
>10
>10
0.40
>10
>10
>10
>10
>10
0.15
>44
>44
>44
>44
>44
>44
>44
>44
>44
>44
>10
9.71
>10
>10
8.56
>10
3.44
>10
>10
8.81
7.07
1.67
>10
>10
4.42
>44
12.38
28.92
21.90
5.57
2.62
>10
>10
>10
>10
>10
0.35
Biochemistry
To verify the mechanism of action of these biologically active
nucleosides, we decided to test the substrate activity and/or
inhibition of polymerases by the corresponding nucleoside tri-
phosphates (NTPs). The parent ribonucleosides 2 were previ-
ously shown[2] to inhibit RNA synthesis, but gemcitabine (4) is
known[4] to inhibit DNA synthesis (its NTP is incorporated by
polymerases but then terminates the chain). Therefore, three
examples of NTPs derived from the title difluororibonucleo-
>10
>10
>10
>10
0.24
0.64
21a
21b
21c
21d
21e
21h
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.76
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.52
1.14
1.13
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sides (11c-TP, 11g-TP, and 11h-TP) were prepared and tested
with bacterial DNA polymerases (as a simple model) and with
human polymerase a.
should be relevant to the cytostatic/cytotoxic effects. The ra-
diolabeled primer was first annealed to a template (TempMonoT
)
and then subjected to primer extension experiment with
human polymerase a (Figure 3). Triphosphates derived from
Enzymatic incorporation of 11c-TP, 11g-TP, and 11h-TP by
bacterial polymerases
The enzymatic incorporation of adenosine derivatives 11c-TP,
11g-TP, and 11h-TP was tested using four different model bac-
terial DNA polymerases (Pwo, KOD XL, Vent(exoꢀ), and
DeepVent(exoꢀ)) and a 31-base-long template (TempPrb4basII), al-
lowing the incorporation of four modifications. The best sub-
strate activities of these modified NTPs were observed with
Vent(exoꢀ) DNA polymerase, which readily and selectively in-
corporated all three modified nucleotides derived from 11 c,
11 g, and 11 h (Figure 2). The formation of full-length oligo-
Figure 3. Denaturing PAGE analysis of PEX products with template
TempMonoT, human DNA polymerase a, and modified nucleoside triphos-
phates 4-TP, 1-TP, 2c-TP, 11c-TP, 11h-TP, and 11g-TP. The experiments were
supplemented with a 32P-radiolabeled primer (p), positive control (+: all nat-
ural dNTPs), and two negative controls (Cꢀ: absence of dCTP; Aꢀ: absence
of dATP).
gemcitabine (4-TP), tubercidin (1-TP), and 7-(2-thienyl)-7-dea-
zaadenosine (2c-TP)[2] were also used in the study to compare
their effects on human polymerase a with the effect of 11c-TP,
11g-TP, and 11h-TP. While nucleotides derived from tubercidin
(1) and 7-thienyltubercidin (2c) were not incorporated into
DNA (and did not show any significant inhibition of the poly-
merase), gemcitabine (4) nucleotide was partly incorporated,
and its incorporation resulted in chain termination. The 7-sub-
stituted 7-deazaadenine difluororibonucleoside triphosphates
11c-TP, 11g-TP, and 11h-TP inhibited the polymerase, yet with
varied potencies. The thienyl and ethynyl nucleotides 11 c and
11 h were incorporated to some extent; however, their incor-
poration led to a stop one base after them. Only traces of the
full-length products were observed. The benzofuryl derivative
11g-TP fully inhibited the polymerase; only a primer band was
observed on the gel.
Figure 2. Denaturing PAGE analysis of PEX products with template
TempPrb4basII, modified 11c-TP, 11g-TP, or 11h-TP and Vent(exoꢀ) DNA poly-
merase. The experiments were supplemented with a 32P-radiolabeled primer
(p), positive control (+: all natural dNTPs), and negative control (Aꢀ: ab-
sence of dATP).
nucleotides with Vent(exoꢀ) DNA polymerase was also con-
firmed by MALDI-TOF analysis (Supporting Information).
KOD XL DNA polymerase efficiently incorporated only 7-thien-
yl- and 7-ethynyl-7-deazaadenine nucleotides 11c-TP and 11 h-
TP, whereas the benzofuryl derivative 11g-TP was not incorpo-
rated. DeepVent(exoꢀ) incorporated only 11c-TP, whereas Pwo
DNA polymerase afforded only a mixture of full-length 31-mer
products together with four-base-shorter oligonucleotide prod-
ucts (figure S1 in Supporting Information). The very good sub-
strate activities of the 7-substituted-7-deazaadenine difluorori-
bonucleoside triphosphates could be used for polymerase syn-
thesis of fluorinated modified DNA.[12,13]
Inhibition of DNA polymerase a activity by 11c-TP, 11g-TP,
and 11h-TP
To determine the effect of 11c-TP, 11g-TP, and 11h-TP on
DNA polymerase a activity, the primer was extended in the
presence of all four natural dNTPs (100 mm) and various con-
centrations of 11c-TP, 11g-TP, and 11h-TP (50–900 mm). The
gel (Figure 4) demonstrates that polymerase a is not inhibited
when 11c-TP and 11h-TP are present up to 100 mm. 11g-TP is
a stronger inhibitor and inhibits the polymerase even at 50 mm.
Therefore, a more detailed study was carried out with 11g-TP
Enzymatic incorporation by human DNA polymerase a
Further studies focused on the incorporation of 11c-TP, 11 g-
TP, and 11h-TP into DNA by human polymerase a, which
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eroaryl-7-deazaadenosines. The
difluororibonucleosides
were
prepared by a low-yielding gly-
cosidation of 6-chloro-7-iodo-7-
deazapurine followed by amina-
tion and cross-couplings. The
fluororibonucleosides were ob-
tained by a longer sequence in-
volving SN2 fluorination of pro-
tected arabinonucleoside inter-
mediates followed by cross-cou-
pling arylations. Some of the
title fluorinated nucleosides 11
and 21 exerted micromolar cyto-
static and anti-HCV activities, sig-
nificantly lower than the (nano-
molar) activities[2] of the parent
ribonucleosides 2. The SAR and
mechanism of action is different
from the ribonucleosides as well.
The NTPs derived from difluoror-
ibonucleosides are inhibitors of
Figure 4. Inhibition of human polymerase a activity by 11c-TP, 11h-TP, and 11g-TP. The reaction mixtures con-
tained all four natural dNTPs (100 mm) together with the modified nucleoside triphosphate 11c-TP, 11h-TP, or
11g-TP (50, 100, 300, or 900 mm). The experiments were supplemented with a 32P-radiolabeled primer (p), positive
control (+: all natural dNTPs) and negative control (Aꢀ: absence of dATP).
human
DNA
polymerase a
at concentrations between 5 and 900 mm (figure S2 in Support-
ing Information). The IC50 value, obtained from the plot
(Figure 5) of primer intensities against the logarithm of 11g-TP
concentrations, is 35 mm. We have not studied the intracellular
(which is most likely the major mode of their action), whereas
they are very good substrates for bacterial DNA polymerases
(e.g., Vent(exoꢀ)) which makes them applicable for the enzy-
matic synthesis of modified fluorinated DNA.
Experimental Section
NMR spectra were recorded using a 500 MHz (1H at 500 MHz, 13C at
125.7 MHz, 19F at 470.3 MHz) spectrometer, in CDCl3 or [D6]DMSO.
Chemical shifts (d) are given in ppm, and coupling constants (J) in
Hz. Melting points were measured on a Kofler block and are uncor-
rected. Both low- and high-resolution MS data were collected
using electrospray ionization. Optical rotations were measured at
258C; [a]D data are given in 10ꢀ1 degcm2 gꢀ1. Reversed-phase high-
performance flash chromatography (HPFC) purifications were done
on a Biotage SP1 apparatus with C18 columns, using a H2O/CH3OH
gradient (0!100%). All final compounds were >98% pure accord-
ing to elemental analysis. 3,5-Di-O-benzoyl-2-deoxy-2,2-difluoro-1-
O-methanesulfonyl-d-erythro-pentofuranose (5) and 6-chloro-7-dea-
zapurine (6) were purchased from Nucleo Chemistry Co. (Shenz-
hen, China). Gemcitabine hydrochloride (4) was purchased from
Sigma–Aldrich.
Figure 5. Inhibition of human polymerase a activity by 11g-TP. Plot of
log(c11g-TP) against the intensity of primer in the denaturing PAGE analysis of
the PEX products with all four natural dNTPs and 11g-TP at an indicated
concentration.
Synthetic oligonucleotides (Prim248short
, , ,
TempPrb4basII TempMonoT
Table 3) were purchased from Sigma–Aldrich. Human DNA poly-
merase a was purchased from Chimerx. DNA polymerases Vent-
(exoꢀ) and DeepVent(exoꢀ), as well as natural nucleoside triphos-
phates (dATP, dCTP, dGTP, dTTP) were purchased from New Eng-
land Biolabs; KOD XL DNA polymerase was obtained from Merck,
and Pwo polymerase was from Peqlab. Streptavidin magnetic parti-
cles were obtained from Roche. All solutions for the PEX experi-
ments were prepared in Milli-Q water.
phosphorylation of nucleosides 11 (but we suppose that it
should be feasible in analogy[2] to ribonucleosides 2), and thus
a plausible mechanism of action is phosphorylation to NTPs
and inhibition of DNA polymerases. However, because we
have not specifically studied the inhibition of other enzymes
(nucleotide metabolism, kinases, etc.) by the nucleosides them-
selves, we cannot rule out other parallel modes of action.
MALDI-TOF spectra were measured on a Reflex IV instrument
(Bruker Daltonics, Germany) with a nitrogen UV laser (l 337 nm).
The measurements were done in reflectron mode by dried droplet
technique, with the mass range up to 100 kDa and resolution
>25000. The matrix consisted of 3-hydroxypicolinic acid (HPA)
Conclusions
In conclusion, we have designed and synthesized novel sugar-
modified fluorinated derivatives of previously reported[2] 7-het-
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ChemMedChem 2013, 8, 832 – 846 837

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Bz), 151.38 (C-7a), 151.60 (CH-2), 153.16 (C-4), 164.52 and
Table 3. Sequences of the primer and templates used in PEX experiment-
165.98 ppm (CO); 19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ116.72 and
s.[a]
~
ꢀ103.35 ppm (2ꢄd, 2ꢄ1F, Jgem =252.4 Hz); IR (ATR): n=2922, 2852,
1740, 1715, 1276, 1254, 1111, 1085, 1061, 1022 cmꢀ1; MS (ESI) m/z
(%): 662 (80) [M+Na], 640 (20) [M+H]; HRMS-ESI [M+H]+ calcd
for C25H18F2N3O5ICl: 639.99422, found: 639.99382.
Prim248short
TempPrb4basII
TempMonoT
5’-CATGGGCGGCATGGG-3’
5’-(bio)-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3’
5’-CTAGCATGAGCTCAGACCCATGCCGCCCATG-3’
[a] Segments that form duplex with the primer are highlighted in bold;
bio=biotin.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
iodopyrrolo[2,3-d]pyrimidine (9): Benzoylated nucleoside
7
(1.98 g, 3.09 mmol) in dioxane (13 mL) and aqueous NH3 (25%
w/w, 12 mL) was stirred in a steel bomb at 1208C for 20 h. After
cooling, the volatiles were removed under reduced pressure and
several times co-evaporated with EtOH. The residual solid was tri-
turated with a boiling solution H2O/CH3OH 4:1 (2 mL) and then
cooled to 48C for 24 h. Solids was filtered off and washed several
times with H2O/CH3OH 4:1 (30 mL) to give a white solid 9 (786 mg,
62%); mp: 247–2558C; [a]D25 = +67.0 cm3 gꢀ1 dmꢀ1 (c=0.106,
/picolinic acid (PA)/ammonium tartrate at a ratio of 9:1:1. The
sample (0.5 mL) and matrix (2 mL) were mixed on target by use of
anchor–chip desk. The crystallized spots were washed once with
0.1% formic acid and once with water.
4-Chloro-7-[3,5-di-O-benzoyl-2-deoxy-2,2-difluoro-b-d-erythro-
pentofuranosyl]-5-iodopyrrolo[2,3-d]pyrimidine (7) and 4-chloro-
7-[3,5-di-O-benzoyl-2-deoxy-2,2-difluoro-a-d-erythro-pentofura-
nosyl]-5-iodopyrrolo[2,3-d]pyrimidine (8): 6-Chloro-7-iodo-7-de-
azapurine (6; 10 g, 35.78 mmol) was dissolved in anhydrous CH3CN
(200 mL), and KOH (3.06 g, 53.68 mmol), TDA-1 (11.46 mL,
35.78 mmol), and mesylate 5 (32.67 g, 71.56 mmol) were added.
The reaction mixture was stirred at 508C for 4 days. Then, the reac-
tion mixture was diluted with EtOAc (150 mL). After adding aque-
ous HCl (1m, 150 mL), a precipitate was formed. This precipitate
was removed by filtration and washed several times with EtOAc
(100 mL). The filtrate was then washed with H2O (150 mL). Organic
phase was dried over MgSO4 and evaporated under reduced pres-
sure. The crude reaction product was purified by column chroma-
tography on silica (hexane/EtOAc 10:1) to obtain crude nucleosides
8 and 7 (2.74 g, 12%) as a mixture of anomers (a/b 1:2). The mix-
ture was purified again by column chromatography on silica
(hexane/EtOAc 12:1!10:1) to give 7 (1.65 g, 8%), and 8 (874 mg,
4%). Both were white solids. Compound 7: mp: 128–1418C;
DMSO); 1H NMR (500 MHz, [D6]DMSO): d=3.66 (dm, 1H, Jgem
12.6 Hz, H-5’a), 3.78 (dm, 1H, Jgem =12.6 Hz, H-5’b), 3.88 (dm, 1H,
=
J
4’,3’ =8.2 Hz, H-4’), 4.39 (m, 1H, H-3’), 5.25 (t, 1H, JOH,5’a =JOH,5’b
=
5.6 Hz, OH-5’), 6.31 (d, 1H, JOH,3’ =6.5 Hz, OH-3’), 6.38 (dd, 1H, J1’,F
=
10.9 and 5.0 Hz, H-1’), 6.78 (bs, 2H, NH2), 7.65 (s, 1H, H-6),
8.14 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=53.56
(C-5), 59.56 (CH2-5’), 68.64 (dd, JC,F =25.7 and 18.6 Hz, CH-3’), 81.07
(d, JC,F =8.4 Hz, CH-4’), 82.43 (dd, JC,F =39.8 and 24.3 Hz, CH-1’),
103.11 (C-4a), 123.14 (dd, JC,F =260.0 and 255.5 Hz, C-2’), 126.76
(CH-6), 150.27 (C-7a), 152.70 (CH-2), 157.51 ppm (C-4); 19F NMR
(470.3 MHz, [D6]DMSO): d=ꢀ113.98 and ꢀ113.00 ppm (2ꢄd, 2ꢄ1F,
~
gem =234.8 Hz); IR (ATR): n=3495, 3394, 2926, 1630, 1558, 1480,
J
1443, 1305, 1203, 1055, 1033 cmꢀ1; MS (ESI) m/z (%): 413 (100)
[M+H], 435 (50) [M+Na]; HRMS-ESI [M+H]+ calcd for
C11H12F2N4O3I: 412.99167, found: 412.99170; Anal. calcd for
C11H11F2N4O3I: C 32.06, H 2.69, N 13.59, found: C 31.80, H 2.61, N
13.22.
1H NMR (500 MHz, CDCl3): d=4.65 (dddd, 1H, J4’,3’ =5.6 Hz, J4’,5’a
4.5 Hz, J4’,5’b =3.5 Hz, J4’,F =1.3 Hz, H-4’), 4.73 (dd, 1H, Jgem =12.4 Hz,
5’a,4’ =4.5 Hz, H-5’a), 4.87 (dd, 1H, Jgem =12.4 Hz, J5’b,4’ =3.6 Hz, H-
=
4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
iodopyrrolo[2,3-d]pyrimidine (10): Compound 10 was prepared
as described for compound 9 from benzoylated nucleoside 8
(838 mg, 1.16 mmol) in dioxane (7 mL) and aqueous NH3 (25%
w/w, 7 mL), to give white solid 6 (420 mg, 88%); mp: 246–2518C;
[a]D25 = +43.6 cm3 gꢀ1 dmꢀ1 (c=0.211, DMSO); 1H NMR (500 MHz,
J
5’b), 5.84 (ddd, 1H, J3’,F =13.4 Hz, J3’,4’ =5.5 Hz, J3’,F =3.7 Hz, H-3’),
6.73 (dd, 1H, J1’,F =11.4 and 6.8 Hz, H-1’), 7.47–7.54 (m, 4H, H-m-
Bz), 7.58 (d, 1H, J6,F =2.8 Hz, H-6), 7.61 and 7.66 (2 m, 2ꢄ1H, H-p-
Bz), 8.08–8.14 (m, 4H, H-o-Bz), 8.66 ppm (s, 1H, H-2); 13C NMR
[D6]DMSO): d=3.57 (ddd, 1H, Jgem =12.4 Hz, J5’a,OH =6.3 Hz, J5’a,4’
4.5 Hz, H-5’a), 3.65 (bdt, 1H, Jgem =12.4 Hz, J5’b,4’ =J5’b,OH =4.3 Hz, H-
5’b), 4.28 (m, 1H, H-4’), 4.43 (m, 1H, H-3’), 5.08 (dd, 1H, JOH,5’a
=
(125.7 MHz, CDCl3): d=54.20 (C-5), 62.55 (CH2-5’), 71.46 (dd, JC,F
=
34.2 and 16.7 Hz, CH-3’), 78.25 (dd, JC,F =4.8 and 2.4 Hz, CH-4’),
82.77 (dd, JC,F =37.9 and 21.5 Hz, CH-1’), 117.43 (C-4a), 120.58 (dd,
=
6.3 Hz, JOH,5’b =5.3 Hz, OH-5’), 6.45 (d, 1H, JOH,3’ =3.7 Hz, OH-3’), 6.57
(dd, 1H, J1’,F =9.4 and 7.6 Hz, H-1’), 6.78 (bs, 2H, NH2), 7.54 (d, 1H,
J
C,F =265.3 and 260.1 Hz, C-2’), 127.88 (C-i-Bz), 128.73 and 128.74
6,F =2.6 Hz, H-6), 8.14 ppm (s, 1H, H-2); 13C NMR (125.7 MHz,
(CH-m-Bz), 129.12 (C-i-Bz), 129.74 and 130.15 (CH-o-Bz), 132.48 (d,
J
J
C,F =4.1 Hz, CH-6), 133.61 and 134.28 (CH-p-Bz), 151.38 (C-7a),
[D6]DMSO): d=53.56 (C-5), 60.27 (CH2-5’), 69.82 (dd, JC,F =26.5 and
17.6 Hz, CH-3’), 82.28 (dd, JC,F =40.5 and 20.5 Hz, CH-1’), 83.99 (d,
151.47 (CH-2), 153.24 (C-4), 164.74 and 166.00 ppm (CO); 19F NMR
(470.3 MHz, [D6]DMSO): d=ꢀ116.57 and ꢀ110.53 ppm (2ꢄd, 2ꢄ1F,
J
C,F =7.1 Hz, CH-4’), 102.88 (C-4a), 123.12 (dd, JC,F =262.3 and
~
gem =246.1 Hz); IR (ATR): n=3146, 2922, 2853, 1718, 1450, 1262,
J
254.8 Hz, C-2’), 127.45 (d, JC,F =2.9 Hz, CH-6), 150.52 (C-7a), 152.68
(CH-2), 157.50 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
ꢀ118.42 and ꢀ109.56 ppm (2ꢄd, 2ꢄ1F, Jgem =234.3 Hz); IR (ATR):
1221, 1106, 1094, 1061, 1033 cmꢀ1; MS (ESI) m/z (%): 662 (100)
[M+Na], 640 (25) [M+H]; HRMS-ESI [M+H]+ calcd for
C25H18F2N3O5ICl: 639.99422, found: 639.99420. Compound 8: mp:
~
n=3473, 3354, 2919, 1630, 1561, 1478, 1446, 1342, 1244, 1057,
1
1030 cmꢀ1; MS (ESI) m/z (%): 413 (100) [M+H], 435 (40) [M+Na];
HRMS-ESI [M+H]+ calcd for C11H12F2N4O3I: 412.99167, found:
412.99171; Anal. calcd for C11H11F2N4O3I: C 32.06, H 2.69, N 13.59,
found: C 31.91, H 2.55, N 13.30.
158–1658C; H NMR (500 MHz, CDCl3): d=4.67–4.74 (m, 2H, H-5’),
5.00 (m, 1H, H-4’), 5.89 (bdt, 1H, J3’,F =12.0 Hz, J3’,F =J3’,4’ =3.8 Hz,
H-3’), 6.94 (dd, 1H, J1’,F =9.0 and 4.2 Hz, H-1’), 7.44–7.57 (m, 4H, H-
m-Bz), 7.60 and 7.67 (2 m, 2ꢄ1H, H-p-Bz), 7.74 (d, 1H, J6,F =1.9 Hz,
H-6), 8.04–8.11 (m, 4H, H-o-Bz), 8.67 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, CDCl3): d=53.87 (C-5), 63.15 (d, JC,F =1.2 Hz, CH2-5’),
72.20 (dd, JC,F =34.4 and 17.3 Hz, CH-3’), 81.46 (dd, JC,F =3.8 and
2.6 Hz, CH-4’), 84.22 (dd, JC,F =42.6 and 22.3 Hz, CH-1’), 117.22 (C-
4a), 122.02 (dd, JC,F =270.4 and 255.3 Hz, C-2’), 127.76 (C-i-Bz),
128.56 and 128.99 (CH-m-Bz), 129.07 (C-i-Bz), 129.83 and 130.07
(CH-o-Bz), 131.98 (d, JC,F =2.4 Hz, CH-6), 133.55 and 134.35 (CH-p-
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(furan-2-yl)pyrrolo[2,3-d]pyrimidine (11a): An argon-purged mix-
ture of compound 9 (150 mg, 0.36 mmol), furan-2-boronic acid
(61.1 mg, 0.55 mmol), Na2CO3 (115.7 mg, 1.09 mmol), Pd(OAc)2
(4.09 mg, 0.018 mmol), and TPPTS (31.0 mg, 0.55 mmol) in H2O/
CH3CN (2:1, 3 mL) was stirred at 1008C for 2 h. After cooling, vola-
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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tiles were removed under reduced pressure, and the residue was
10.7 and 5.2 Hz, H-1’), 7.17–7.21 (m, 2H, H-3,4-thienyl), 7.59 (m, 1H,
H-5-thienyl), 7.60 (s, 1H, H-6), 8.20 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=59.51 (CH2-5’), 68.63 (dd, JC,F =25.1 and
19.2 Hz, CH-3’), 81.05 (d, JC,F =7.7 Hz, CH-4’), 82.44 (dd, JC,F =39.3
and 24.6 Hz, CH-1’), 100.43 (C-4a), 109.71 (C-5), 121.47 (CH-6),
123.27 (bdd, JC,F =259.6 and 256.4 Hz, C-2’), 126.27 (CH-5-thienyl),
126.87 (CH-3-thienyl), 128.55 (CH-4-thienyl), 135.07 (C-2-thienyl),
150.83 (C-7a), 152.77 (CH-2), 157.56 ppm (C-4); 19F NMR (470.3 MHz,
purified by reversed-phase HPFC on C18 (0!100% CH3OH in H2O)
to give compound 11 a (84 mg, 65%) as a beige solid after recrys-
25
tallization
(H2O/CH3OH
4:1);
mp:
189–1918C;
[a]D =
ꢀ3.8 cm3 gꢀ1 dmꢀ1 (c=0.236, DMSO); H NMR (500 MHz, [D6]DMSO):
1
d=3.69 (ddd, 1H, Jgem =12.6 Hz, J5’a,OH =6.0 Hz, J5’a,4’ =4.2 Hz, H-
5’a), 3.81 (dm, 1H, Jgem =12.6 Hz, H-5’b), 3.91 (ddd, 1H, J4’,3’
=
8.1 Hz, J4’,5’a =4.2 Hz, J4’,5’b =2.8 Hz, H-4’), 4.43 (m, 1H, H-3’), 5.27 (t,
1H, JOH,5’a =JOH,5’b =5.7 Hz, OH-5’), 6.33 (d, 1H, JOH,3’ =6.5 Hz, OH-3’),
6.47 (dd, 1H, J1’,F =10.9 and 5.2 Hz, H-1’), 6.62 (dd, 1H, J4,3 =3.3 Hz,
[D6]DMSO): d=ꢀ113.92 and ꢀ113.05 ppm (2ꢄd, 2ꢄ1F, Jgem
=
~
233.7 Hz); IR (ATR): n=3337, 3139, 1632, 1588, 1546, 1464, 1311,
1207, 1077, 1057 cmꢀ1; MS (ESI) m/z (%): 369 (100) [M+H], 391
(23) [M+Na]; HRMS-ESI [M+H]+ calcd for C15H15F2N4O3S:
J
4,5 =1.9 Hz, H-4-furyl), 6.70 (dd, 1H, J3,4 =3.3 Hz, J3,5 =0.9 Hz, H-3-
furyl), 7.00 (bs, 2H, NH2), 7.80 (dd, 1H, J5,4 =1.9 Hz, J5,3 =0.8 Hz, H-
5-furyl), 7.81 (bs, 1H, H-6), 8.18 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=59.71 (CH2-5’), 68.84 (dd, JC,F =25.3 and
19.0 Hz, CH-3’), 81.13 (d, JC,F =8.0 Hz, CH-4’), 82.44 (dd, JC,F =40.0
and 24.4 Hz, CH-1’), 99.11 (C-4a), 105.92 (CH-3-furyl), 107.36 (C-5),
112.13 (CH-4-furyl), 119.92 (CH-6), 123.20 (dd, JC,F =260.2 and
255.1 Hz, C-2’), 142.49 (CH-5-furyl), 148.25 (C-2-furyl), 151.10 (C-7a),
152.85 (CH-2), 157.54 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO):
d=ꢀ113.95 and ꢀ112.95 ppm (2ꢄd, 2ꢄ1F, Jgem =234.1 Hz); IR
369.08274,
found:
369.08274;
Anal.
calcd
for
C15H14F2N4O3S·0.4CH3OH·0.8H2O: C 46.76, H 4.38, N 14.16, found: C
46.63, H 4.15, N 13.94.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(thiophen-3-yl)pyrrolo[2,3-d]pyrimidine (11d): Compound 11 d
was prepared as described for compound 11 a from compound 9
(150 mg, 0.36 mmol) and thiophene-3-boronic acid (69.9 mg,
0.55 mmol), to give compound 11 d (81 mg, 60%) as a light-brown
solid after recrystallization (H2O/CH3OH 4:1); mp: 116–1228C;
[a]D25 = +1.6 cm3 gꢀ1 dmꢀ1 (c=0.189, DMSO); 1H NMR (500 MHz,
~
(ATR): n=3487, 3383, 3128, 1634, 1557, 1487, 1457, 1318, 1203,
1071, 1041 cmꢀ1; MS (ESI) m/z (%): 353 (100) [M+H], 375 (40) [M+
Na]; HRMS-ESI [M+H]+ calcd for C15H15F2N4O4: 353.10559, found:
353.10559; Anal. calcd for C15H14F2N4O4·0.75H2O: C 49.25, H 4.27, N
15.32, found: C 49.58, H 4.15, N 14.95.
[D6]DMSO): d=3.67 (ddd, 1H, Jgem =12.6 Hz, J5’a,OH =5.9 Hz, J5’a,4’
=
4.2 Hz, H-5’a), 3.79 (bdm, 1H, Jgem =12.6 Hz, H-5’b), 3.90 (m, 1H, H-
4’), 4.44 (m, 1H, H-3’), 5.23 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’),
6.31 (bs, 2H, NH2), 6.32 (d, 1H, JOH,3’ =6.3 Hz, OH-3’), 6.47 (dd, 1H,
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(furan-3-yl)pyrrolo[2,3-d]pyrimidine (11b): Compound 11 b was
prepared as described for compound 11 a from compound 9
(150 mg, 0.36 mmol) and furan-3-boronic acid (61.1 mg,
J
1’,F =11.1 and 5.2 Hz, H-1’), 7.27 (dd, 1H, J4,5 =4.9 Hz, J4,2 =1.3 Hz,
H-4-thienyl), 7.53 (s, 1H, H-6), 7.56 (dd, 1H, J2,5 =2.9 Hz, J2,4 =1.3 Hz,
H-2-thienyl), 7.72 (dd, 1H, J5,4 =4.9 Hz, J5,2 =2.9 Hz, H-5-thienyl),
8.18 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=59.66
0.55 mmol), to give compound 11 b (71 mg, 55%) as a white solid
25
after recrystallization (H2O/CH3OH 4:1); mp: 106–1108C; [a]D
=
(CH2-5’), 68.82 (dd, JC,F =24.9 and 19.3 Hz, CH-3’), 81.01 (d, JC,F
=
0 cm3 gꢀ1 dmꢀ1 (c=0.157, DMSO); 1H NMR (500 MHz, [D6]DMSO):
d=3.67 (ddd, 1H, Jgem =12.6 Hz, J5’a,OH =6.0 Hz, J5’a,4’ =4.2 Hz, H-
5’a), 3.79 (dm, 1H, Jgem =12.6 Hz, H-5’b), 3.89 (m, 1H, H-4’), 4.43 (m,
1H, H-3’), 5.23 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’), 6.32 (d, 1H,
7.8 Hz, CH-4’), 82.39 (dd, JC,F =39.4 and 24.6 Hz, CH-1’), 100.61
(C-4a), 112.18 (C-5), 120.56 (CH-6), 122.66 (CH-2-thienyl), 123.29
(dd, JC,F =259.4 and 255.7 Hz, C-2’), 127.73 (CH-5-thienyl), 128.58
(CH-4-thienyl), 134.39 (C-3-thienyl), 150.85 (C-7a), 152.50 (CH-2),
157.68 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ113.85 and
J
OH,3’ =6.5 Hz, OH-3’), 6.37 (bs, 2H, NH2), 6.45 (dd, 1H, J1’,F =11.1
and 5.3 Hz, H-1’), 6.71 (dd, 1H, J4,5 =1.8 Hz, J4,2 =0.9 Hz, H-4-furyl),
7.48 (s, 1H, H-6), 7.82 (t, 1H, J5,2 =J5,4 =1.7 Hz, H-5-furyl), 7.86 (dd,
1H, J2,5 =1.6 Hz, J2,4 =0.9 Hz, H-2-furyl), 8.17 ppm (s, 1H, H-2);
~
ꢀ112.94 ppm (2ꢄd, 2ꢄ1F, Jgem =233.6 Hz); IR (ATR): n=3419, 3327,
3113, 1632, 1592, 1549, 1463, 1311, 1210, 1066, 1032 cmꢀ1. (ESI)
m/z (%): 369 (100) [M+H], 391 (40) [M+Na]; HRMS-ESI [M+H]+
calcd for C15H15F2N4O3S: 369.08274, found: 369.08272; Anal. calcd
for C15H14F2N4O3S: C 48.91, H 3.83, N 15.21, found: C 48.66, H 3.82,
N 14.94.
13C NMR (125.7 MHz, [D6]DMSO): d=59.71 (CH2-5’), 68.87 (dd, JC,F
=
25.4 and 18.6 Hz, CH-3’), 81.02 (d, JC,F =8.3 Hz, CH-4’), 82.38 (dd,
C,F =39.3 and 24.3 Hz, CH-1’), 100.82 (C-4a), 107.55 (C-5), 111.66
J
(CH-4-furyl), 118.34 (C-3-furyl), 120.49 (CH-6), 123.27 (dd, JC,F =260.0
and 255.6 Hz, C-2’), 140.12 (CH-2-furyl), 144.50 (CH-5-furyl), 151.01
(C-7a), 152.55 (CH-2), 157.77 ppm (C-4); 19F NMR (470.3 MHz,
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
phenyl-pyrrolo[2,3-d]pyrimidine (11e): Compound 11 e was pre-
pared as described for compound 11 a from compound 9 (140 mg,
0.34 mmol) and phenylboronic acid (62.2 mg, 0.51 mmol), to give
compound 11 e (87 mg, 71%) as a white solid after recrystallization
(H2O/CH3OH 4:1); mp: 122–1248C; [a]D25 =ꢀ1.8 cm3 gꢀ1 dmꢀ1 (c=
0.224, DMSO); 1H NMR (500 MHz, [D6]DMSO): d=3.67 (ddd, 1H,
[D6]DMSO): d=ꢀ113.91 and ꢀ112.89 ppm (2ꢄd, 2ꢄ1F, Jgem
=
~
233.5 Hz); IR (ATR): n=3490, 3378, 3163, 1626, 1563, 1475, 1457,
1307, 1210, 1086, 1035 cmꢀ1; MS (ESI) m/z (%): 353 (100) [M+H],
375 (25) [M+Na]; HRMS-ESI [M+H]+ calcd for C15H15F2N4O4:
353.10559, found: 353.10554; Anal. calcd for C15H14F2N4O4: C 51.14,
H 4.01, N 15.90, found: C 51.03, H 4.10, N 15.76.
J
J
J
gem =12.6 Hz, J5’a,OH =6.0 Hz, J5’a,4’ =4.1 Hz, H-5’a), 3.79 (bdm, 1H,
gem =12.6 Hz, H-5’b), 3.91 (bddd, 1H, J4’,3’ =8.3 Hz, J4’,5’a =4.0 Hz,
4’,5’b =2.6 Hz, H-4’), 4.46 (m, 1H, H-3’), 5.24 (t, 1H, JOH,5’a =JOH,5’b =
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(thiophen-2-yl)pyrrolo[2,3-d]pyrimidine (11c): Compound 11 c
was prepared as described for compound 11 a from compound 9
(150 mg, 0.36 mmol) and thiophene-2-boronic acid (69.9 mg,
5.6 Hz, OH-5’), 6.25 (bs, 2H, NH2), 6.32 (d, 1H, JOH,3’ =5.4 Hz, OH-3’),
6.49 (dd, 1H, J1’,F =10.9 and 5.3 Hz, H-1’), 7.39 (m, 1H, H-p-Ph),
7.47–7.52 (m, 4H, H-o,m-Ph), 7.53 (s, 1H, H-6), 8.20 ppm (s, 1H, H-
2); 13C NMR (125.7 MHz, [D6]DMSO): d=59.66 (CH2-5’), 68.81 (dd,
0.55 mmol), to give compound 11 c (71 mg, 53%) as a white solid
25
after recrystallization (H2O/CH3OH 4:1); mp: 112–1148C; [a]D
=
JC,F =25.0 and 19.2 Hz, CH-3’), 81.03 (d, JC,F =8.1 Hz, CH-4’), 82.44
ꢀ6.7 cm3 gꢀ1 dmꢀ1 (c=0.105, DMSO); H NMR (500 MHz, [D6]DMSO):
d=3.67 (bddd, 1H, Jgem =12.7 Hz, J5’a,OH =5.9 Hz, J5’a,4’ =4.0 Hz, H-
5’a), 3.79 (bdm, 1H, Jgem =12.7 Hz, H-5’b), 3.90 (m, 1H, H-4’), 4.45
(m, 1H, H-3’), 5.27 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’), 6.32 (bd,
(dd, JC,F =39.0 and 24.9 Hz, CH-1’), 100.31 (C-4a), 117.53 (C-5),
120.67 (CH-6), 123.35 (dd, JC,F =259.7 and 255.4 Hz, C-2’), 127.39
(CH-p-Ph), 128.66 and 129.27 (CH-o,m-Ph), 134.20 (C-i-Ph), 151.14
(C-7a), 152.49 (CH-2), 157.62 ppm (C-4); 19F NMR (470.3 MHz,
1
1H, JOH,3’ =6.0 Hz, OH-3’), 6.41 (bs, 2H, NH2), 6.47 (bdd, 1H, J1’,F
=
[D6]DMSO): d=ꢀ113.81 and ꢀ112.92 ppm (2ꢄd, 2ꢄ1F, Jgem
=
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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~
~
233.9 Hz); IR (ATR): n=3437, 3348, 3156, 1630, 1582, 1537, 1462,
n=3453, 3301, 3138, 1650, 1573, 1560, 1454, 1318, 1209, 1098,
1076 cmꢀ1; MS (ESI) m/z (%): 403 (100) [M+H], 425 (60) [M+Na];
HRMS-ESI [M+H]+ calcd for C19H17F2N4O4: 403.12124, found:
413.12109; Anal. calcd for C19H16F2N4O4·0.35H2O: C 55.84, H 4.12, N
13.71, found: C 56.06, H 4.05, N 13.37.
1307, 1211, 1062, 1035 cmꢀ1; MS (ESI) m/z (%): 363 (100) [M+H],
385 (20) [M+Na]; HRMS-ESI [M+H]+ calcd for C17H17F2N4O3:
363.12632, found: 363.12628; Anal. calcd for C17H16F2N4O3·1.2H2O:
C 53.18, H 4.83, N 14.59, found: C 53.35, H 4.69, N 14.3.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(naphtalen-2-yl)pyrrolo[2,3-d]pyrimidine (11 f): Compound 11 f
was prepared as described for compound 11 a from compound 9
(150 mg, 0.36 mmol) and naphtalene-2-boronic acid (93.9 mg,
0.55 mmol). Crude product was purified by column chromatogra-
phy on silica in 3% CH3OH in CHCl3, and by reversed-phase HPFC
on C18 (0!100% CH3OH in H2O) to give compound 11 f (110 mg,
71%) as a white solid after recrystallization (H2O/CH3OH 4:1); mp:
143–1468C; [a]D25 =0 cm3 gꢀ1 dmꢀ1 (c=0.211, DMSO); 1H NMR
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
[(trimethylsilyl)ethynyl]-pyrrolo[2,3-d]pyrimidine (12): An argon-
purged mixture of derivative 9 (200 mg, 0.49 mmol), PdCl2(PPh3)2
(17 mg, 0.024 mmol), CuI (9.22 mg, 0.049 mmol), trimethylsilylace-
tylene (0.68 mL, 4.9 mmol) and triethylamine (200 mL) was stirred in
DMF (800 mL) at room temperature for 18 h. Volatiles were re-
moved under reduced pressure and crude product is purified twice
by column chromatography on silica in 3% CH3OH in CHCl3. The
product was then purified by reversed-phase HPFC on C18 (0!
100% CH3OH in H2O) to give trimethylsilylethynyl derivative 12
(139 mg, 75%) as a white crystalline solid; mp: 188–1868C;
1H NMR (500 MHz, [D6]DMSO): d=0.25 (s, 3ꢄ3H, CH3Si), 3.67 (ddd,
1H, Jgem =12.7 Hz, J5’a,OH =6.1 Hz, J5’a,4’ =4.0 Hz, H-5’a), 3.79 (bddd,
1H, Jgem =12.7 Hz, J5’b,OH =5.0 Hz, J5’b,4’ =2.5 Hz, H-5’b), 3.89 (ddd,
1H, J4’,3’ =8.3 Hz, J4’,5’a =4.0 Hz, J4’,5’b =2.5 Hz, H-4’), 4.40 (m, 1H, H-
(500 MHz, [D6]DMSO): d=3.68 (ddd, 1H, Jgem =12.7 Hz, J5’a,OH
=
6.0 Hz, J5’a,4’ =4.1 Hz, H-5’a), 3.81 (dm, 1H, Jgem =12.6 Hz, H-5’b),
3.92 (bddd, 1H, J4’,3’ =8.1 Hz, J4’,5’a =4.0 Hz, J4’,5’b =2.7 Hz, H-4’), 4.49
(m, 1H, H-3’), 5.25 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’), 6.32 (bs,
2H, NH2), 6.34 (d, 1H, JOH,3’ =6.3 Hz, OH-3’), 6.53 (dd, 1H, J1’,F =11.0
and 5.5 Hz, H-1’), 7.51–7.59 (m, 2H, H-6,7-naphthyl), 7.642 (s, 1H,
H-6), 7.644 (dd, 1H, J3,4 =8.4 Hz, J3,1 =1.8 Hz, H-3-naphthyl), 7.95–
8.00 (m, 2H, H-5,8-naphthyl), 8.00 (bd, 1H, J1,3 =1.9 Hz, H-1-naph-
thyl), 8.04 (bd, 1H, J4,3 =8.5 Hz, H-4-naphthyl), 8.22 ppm (s, 1H,
H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=59.69 (CH2-5’), 68.88 (dd,
3’), 5.26 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’), 6.33 (d, 1H, JOH,3’
=
6.3 Hz, OH-3’), 6.39 (bdd, 1H, J1’,F =10.1 and 5.4 Hz, H-1’), 7.81 (s,
1H, H-6), 8.17 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO):
d=ꢀ0.04 (CH3-Si), 59.57 (CH2-5’), 68.57 (dd, JC,F =24.1 and 20.0 Hz,
CH-3’), 81.19 (d, JC,F =7.7 Hz, CH-4’), 82.61 (dd, JC,F =37.9 and
26.5 Hz, CH-1’), 95.95 (C-5), 97.60 (CꢁCSi), 98.64 (CꢁCSi), 102.08
(C-4a), 123.14 (bt, JC,F =257.6 Hz, C-2’), 126.90 (CH-6), 149.70 (C-7a),
153.63 (CH-2), 157.85 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO):
d=ꢀ113.91 and ꢀ113.24 ppm (2ꢄd, 2ꢄ1F, Jgem =234.2 Hz); IR
J
C,F =25.4 and 19.4 Hz, CH-3’), 81.04 (d, JC,F =7.9 Hz, CH-4’), 82.47
(dd, JC,F =39.8 and 24.4 Hz, CH-1’), 100.41 (C-4a), 117.56 (C-5),
121.00 (CH-6), 123.35 (bdd, JC,F =259.6 and 255.2 Hz, C-2’), 126.22
(CH-6-naphthyl), 126.77 (CH-7-naphthyl), 126.99 (CH-1-naphthyl),
127.07 (CH-3-naphthyl), 127.86 and 128.07 (CH-5,8-naphthyl),
128.76 (CH-4-naphthyl), 131.64 (C-2-naphthyl), 132.14 (C-4a-naph-
thyl), 133.41 (C-8a-naphthyl), 151.29 (C-7a), 152.54 (CH-2),
157.72 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ113.77 and
~
(ATR): n=3505, 3395, 2959, 2159, 1636, 1577, 1248, 1204, 1070,
1042, 1019 cmꢀ1; MS (ESI) m/z (%): 383 (100) [M+H], 405 (40) [M+
Na]; HRMS-ESI [M+H]+ calcd for C16H21F2N4O3Si: 383.13455, found:
383.13459.
~
ꢀ112.80 ppm (2ꢄd, 2ꢄ1F, Jgem =233.3 Hz); IR (ATR): n=3441, 3311,
3201, 1627, 1583, 1564, 1472, 1308, 1202, 1066 cmꢀ1; MS (ESI) m/z
(%): 413 (100) [M+H], 435 (13) [M+Na]; HRMS-ESI [M+H]+ calcd
for C21H19F2N4O3: 413.14197, found: 413.14195; Anal. calcd for
C21H18F2N4O3·1.15H2O: C 58.24, H 4.72, N 12.94, found: C 58.47, H
4.57, N 12.61.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
ethynyl-pyrrolo[2,3-d]pyrimidine (11h): A mixture of the silylated
compound 12 (120 mg, 0.31 mmol) and K2CO3 (43.4 mg,
0.31 mmol) in CH3OH (5 mL) was stirred at room temperature for
1 h. The crude product was purified by column chromatography
on silica in 3% CH3OH in CHCl3. Then, the product was purified by
reversed-phase HPFC on C18 (0!100% CH3OH in H2O) to give
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(benzofuran-2-yl)pyrrolo[2,3-d]pyrimidine (11g): Compound 11 g
was prepared as described for compound 11 a from compound 9
(150 mg, 0.36 mmol) and 2-benzofurylboronic acid (88.4 mg,
0.55 mmol). The crude product was purified by column chromatog-
raphy on silica in 3% CH3OH in CHCl3, and by reversed-phase HPFC
on C18 (0!100% CH3OH in H2O), to give compound 11 g (57 mg,
39%) as a white solid after recrystallization (H2O/CH3OH 4:1); mp:
223–2268C; [a]D25 =ꢀ6.8 cm3 gꢀ1 dmꢀ1 (c=0.222, DMSO); 1H NMR
(500 MHz, [D6]DMSO): d=3.72 (m, 1H, H-5’a), 3.84 (m, 1H, H-5’b),
compound 11 h (58.1 mg, 60%) as a white crystalline solid after re-
25
crystallization (H2O/CH3OH 4:1); mp: 219–2258C; [a]D
=
+2.7 cm3 gꢀ1 dmꢀ1 (c=0.219, DMSO); 1H NMR (500 MHz,
[D6]DMSO): d=3.67 (ddd, 1H, Jgem =12.7 Hz, J5’a,OH =6.0 Hz, J5’a,4’
4.0 Hz, H-5’a), 3.79 (dm, 1H, Jgem =12.7 Hz, H-5’b), 3.89 (ddd, 1H,
4’,3’ =8.3 Hz, J4’,5’a =3.9 Hz, J4’,5’b =2.5 Hz, H-4’), 4.34 (s, 1H, CH), 4.41
(m, 1H, H-3’), 5.30 (t, 1H, JOH,5’a =JOH,5’b =5.5 Hz, OH-5’), 6.35 (d, 1H,
OH,3’ =6.3 Hz, OH-3’), 6.39 (dd, 1H, J1’,F =9.9 and 5.6 Hz, H-1’), 6.77
=
J
3.95 (m, 1H, H-4’), 4.48 (m, 1H, H-3’), 5.32 (bt, 1H, JOH,5’a =JOH,5’b
5.6 Hz, OH-5’), 6.37 (d, 1H, JOH,3’ =6.5 Hz, OH-3’), 6.52 (bdd, 1H,
1’,F =10.6 and 5.0 Hz, H-1’), 7.10 (bs, 2H, NH2), 7.16 (s, 1H, H-3-ben-
=
J
(bs, 2H, NH2), 7.81 (s, 1H, H-6), 8.16 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=59.54 (CH2-5’), 68.53 (dd, JC,F =23.5 and
20.5 Hz, CH-3’), 76.91 (CꢁCH), 81.17 (d, JC,F =7.0 Hz, CH-4’), 82.61
(dd, JC,F =37.7 and 26.5 Hz, CH-1’), 83.88 (CꢁCH), 95.35 (C-5),
102.12 (C-4a), 123.18 (bdd, JC,F =258.8 and 256.8 Hz, C-2’), 126.95
(CH-6), 149.76 (C-7a), 153.59 (CH-2), 157.80 ppm (C-4); 19F NMR
(470.3 MHz, [D6]DMSO): d=ꢀ113.99 and ꢀ113.33 ppm (2ꢄd, 2ꢄ1F,
J
zofuryl), 7.26–7.34 (m, 2H, H-5,6-benzofuryl), 7.64–7.71 (m, 2H,
H-3,4-benzofuryl), 8.07 (s, 1H, H-6), 8.23 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=59.73 (CH2-5’), 68.83 (dd, JC,F =25.8 and
18.9 Hz, CH-3’), 81.24 (d, JC,F =8.2 Hz, CH-4’), 82.56 (dd, JC,F =40.1
and 24.2 Hz, CH-1’), 99.23 (C-4a), 102.39 (CH-3-benzofuryl), 106.79
(C-5), 111.37 (CH-7-benzofuryl), 120.95 (CH-4-benzofuryl), 122.16
(CH-6), 123.24 (dd, JC,F =260.4 and 255.7 Hz, C-2’), 123.71 (CH-5-
benzofuryl), 124.25 (CH-6-benzofuryl), 128.91 (C-3a-benzofuryl),
150.71 (C-2-benzofuryl), 151.34 (C-7a), 153.07 (CH-2), 154.07 (C-7a-
benzofuryl), 157.60 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
ꢀ113.93 and ꢀ112.89 ppm (2ꢄd, 2ꢄ1F, Jgem =233.9 Hz); IR (ATR):
~
gem =233.8 Hz); IR (ATR): n=3495, 3448, 3394, 3330, 3165, 1632,
J
1576, 1450, 1303, 1180, 1029, 1012 cmꢀ1; MS (ESI) m/z (%): 311
(100) [M+H], 333 (50) [M+Na]; HRMS-ESI [M+H]+ calcd for
C13H13F2N4O3: 311.09502, found: 311.09497; Anal. calcd for
C13H13F2N4O3: C 50.33, H 3.90, N 18.06, found: C 50.26, H 3.82, N
17.74.
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
(furan-2-yl)pyrrolo[2,3-d]pyrimidine (13a): Compound 13a was
prepared as described for compound 11 a from compound 10
(146 mg, 0.36 mmol) and furan-2-boronic acid (61.1 mg,
[D6]DMSO): d=3.59 (dd, 1H, Jgem =12.5 Hz, J5’a,4’ =4.6 Hz, H-5’a),
3.67 (bdd, 1H, Jgem =12.5 Hz, J5’b,4’ =3.1 Hz, H-5’b), 4.30 (m, 1H, H-
4’), 4.46 (m, 1H, H-3’), 5.12 (bs, 1H, OH-5’), 6.20–6.80 (m, 3H, NH2,
OH-3’), 6.66 (bt, 1H, J1’,F =8.6 Hz, H-1’), 7.17–7.21 (m, 2H, H-3,4-
thienyl), 7.46 (d, 1H, J6,F =2.7 Hz, H-6), 7.59 (m, 1H, H-5-thienyl),
8.20 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.34
0.55 mmol), to give compound 13a (49 mg, 38%) as a beige solid
25
after recrystallization (H2O/CH3OH 4:1); mp: 243–2488C; [a]D
=
+55.3 cm3 gꢀ1 dmꢀ1 (c=0.170, DMSO); 1H NMR (500 MHz,
[D6]DMSO): d=3.60 (ddd, 1H, Jgem =12.4 Hz, J5’a,OH =6.2 Hz, J5’a,4’
(CH2-5’), 69.91 (dd, JC,F =26.7 and 18.1 Hz, CH-3’), 82.28 (dd, JC,F =
=
40.6 and 20.3 Hz, CH-1’), 84.05 (d, JC,F =7.0 Hz, CH-4’), 100.23 (C-4a),
109.81 (C-5), 122.11 (d, JC,F =2.6 Hz, CH-6), 123.27 (dd, JC,F =262.2
and 254.5 Hz, C-2’), 126.30 (CH-5-thienyl), 126.92 (CH-3-thienyl),
128.59 (CH-4-thienyl), 135.02 (C-2-thienyl), 151.14 (C-7a), 152.81
(CH-2), 157.59 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
ꢀ118.34 and ꢀ109.22 ppm (2ꢄd, 2ꢄ1F, Jgem =234.6 Hz); IR (ATR):
4.4 Hz, H-5’a), 3.69 (dm, 1H, Jgem =12.4 Hz, H-5’b), 4.33 (m, 1H, H-
4’), 4.48 (m, 1H, H-3’), 5.12 (dd, 1H, JOH,5’a =6.2 Hz, JOH,5’b =5.2 Hz,
OH-5’), 6.44 (d, 1H, JOH,3’ =5.4 Hz, OH-3’), 6.62 (dd, 1H, J4,3 =3.4 Hz,
J
4,5 =1.9 Hz, H-4-furyl), 6.66 (dd, 1H, J1’,F =10.4 and 7.0 Hz, H-1’),
6.78 (dd, 1H, J3,4 =3.3 Hz, J3,5 =0.8 Hz, H-3-furyl), 7.02 (bs, 2H, NH2),
7.73 (d, 1H, J6,F =2.8 Hz, H-6), 7.80 (dd, 1H, J5,4 =1.9 Hz, J5,3 =0.8 Hz,
H-5-furyl), 8.17 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO):
d=60.31 (CH2-5’), 69.76 (dd, JC,F =26.0 and 18.1 Hz, CH-3’), 82.19
(dd, JC,F =40.0 and 19.9 Hz, CH-1’), 83.78 (d, JC,F =7.6 Hz, CH-4’),
98.89 (C-4a), 105.93 (CH-3-furyl), 107.41 (C-5), 112.16 (CH-4-furyl),
120.50 (bd, JC,F =2.0 Hz, CH-6), 123.09 (dd, JC,F =261.8 and 255.8 Hz,
C-2’), 142.38 (CH-5-furyl), 148.26 (C-2-furyl), 151.33 (C-7a), 152.80
(CH-2), 157.52 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
ꢀ119.08 and ꢀ110.75 ppm (2ꢄd, 2ꢄ1F, Jgem =232.7 Hz); IR (ATR):
n=3482, 3372, 3242, 1616, 1549, 1459, 1190, 1065, 1041 cmꢀ1; MS
~
(ESI) m/z (%): 369 (100) [M+H], 391 (60) [M+Na]; HRMS-ESI [M+
H]+ calcd for C15H15F2N4O3S: 369.08274, found: 369.08278; Anal.
calcd for C15H14F2N4O3S: C 48.91, H 3.83, N 15.21, found: C 48.55, H
3.86, N 14.99.
4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
(thiophen-3-yl)pyrrolo[2,3-d]pyrimidine (13d): Compound 13d
was prepared as described for compound 11 a from compound 10
(140 mg, 0.34 mmol) and thiophene-3-boronic acid (65.3 mg,
0.51 mmol). The crude product was purified by column chromatog-
raphy on silica in 3% CH3OH in CHCl3, and by reversed-phase HPFC
on C18 (0!100% CH3OH in H2O), to give compound 13d (81 mg,
65%) as a white solid after recrystallization (H2O/CH3OH 4:1); mp:
~
n=3470, 3345, 3186, 1621, 1571, 1557, 1456, 1300, 1229, 1067,
1055 cmꢀ1; MS (ESI) m/z (%): 353 (100) [M+H], 375 (20) [M+Na];
HRMS-ESI [M+H]+ calcd for C15H15F2N4O4: 353.10559, found:
353.10558; Anal. calcd for C15H14F2N4O4.0.5 CH3OH: C 50.55, H 4.38,
N 15.21, found: C 50.38, H 4.12, N 14.96.
1
154–1558C; [a]D25 = +76.6 cm3 gꢀ1 dmꢀ1 (c=0.197, DMSO); H NMR
4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
(furan-3-yl)pyrrolo[2,3-d]pyrimidine (13b): Compound 13b was
prepared as described for compound 11 a from compound 10
(144 mg, 0.35 mmol) and furan-3-boronic acid (63.9 mg,
(500 MHz, [D6]DMSO): d=3.59 (ddd, 1H, Jgem =12.4 Hz, J5’a,OH
6.3 Hz, J5’a,4’ =4.4 Hz, H-5’a), 3.68 (bdt, 1H, Jgem =12.4 Hz, J5’b,OH
=
=
J
J
J
J
5’b,4’ =4.3 Hz, H-5’b), 4.29 (bdddd, 1H, J4’,3’ =7.3 Hz, J4’,5’a =4.4 Hz,
4’,5’b =3.4 Hz, J4’,F =1.3 Hz, H-4’), 4.46 (m, 1H, H-3’), 5.11 (dd, 1H,
OH,5’a =6.2 Hz, JOH,5’b =5.3 Hz, OH-5’), 6.32 (bs, 2H, NH2), 6.46 (d, 1H,
OH,3’ =3.6 Hz, OH-3’), 6.67 (dd, 1H, J1’,F =9.6 and 7.6 Hz, H-1’), 7.30
0.52 mmol), to give compound 13b (75 mg, 61%) as a white solid
25
after recrystallization (H2O/CH3OH 4:1); mp: 102–1058C; [a]D
=
+62.8 cm3 gꢀ1 dmꢀ1 (c=0.121, DMSO); 1H NMR (500 MHz,
[D6]DMSO): d=3.59 (ddd, 1H, Jgem =12.4 Hz, J5’a,OH =6.3 Hz, J5’a,4’
(dd, 1H, J4,5 =4.9 Hz, J4,2 =1.3 Hz, H-4-thienyl), 7.43 (d, 1H, J6,F =
=
2.9 Hz, H-6), 7.58 (dd, 1H, J2,5 =2.9 Hz, J2,4 =1.3 Hz, H-2-thienyl),
7.72 (dd, 1H, J5,4 =4.9 Hz, J5,2 =2.9 Hz, H-5-thienyl), 8.18 ppm (s, 1H,
H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.36 (CH2-5’), 69.90 (dd,
4.4 Hz, H-5’a), 3.67 (bdt, 1H, Jgem =12.4 Hz, J5’b,4’ =J5’b,OH =4.3 Hz,
H-5’b), 4.27 (bdddd, 1H, J4’,3’ =7.3 Hz, J4’,5’a =4.3 Hz, J4’,5’a =3.2 Hz,
J
J
4’,F =1.3 Hz, H-4’), 4.46 (m, 1H, H-3’), 5.10 (dd, 1H, JOH,5’a =6.2 Hz,
OH,5’b =5.4 Hz, OH-5’), 6.37 (bs, 2H, NH2), 6.44 (d, 1H, JOH,3’ =6.2 Hz,
JC,F =26.5 and 17.9 Hz, CH-3’), 81.18 (dd, JC,F =40.6 and 20.3 Hz, CH-
1’), 83.88 (d, JC,F =7.3 Hz, CH-4’), 100.43 (C-4a), 112.26 (C-5), 121.21
(d, JC,F =2.9 Hz, CH-6), 122.70 (CH-2-thienyl), 123.27 (dd, JC,F =261.6
and 254.6 Hz, C-2’), 127.74 (CH-5-thienyl), 128.61 (CH-4-thienyl),
134.34 (C-3-thienyl), 151.13 (C-7a), 152.51 (CH-2), 157.70 ppm (C-4);
19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ118.46 and ꢀ109.47 ppm
OH-3’), 6.65 (dd, 1H, J1’,F =10.1 and 7.4 Hz, H-1’), 6.74 (dd, 1H, J4,5
1.8 Hz, J4,2 =0.9 Hz, H-4-furyl), 7.38 (d, 1H, J6,F =2.9 Hz, H-6), 7.82 (t,
1H, J5,2 =J5,4 =1.7 Hz, H-5-furyl), 7.88 (dd, 1H, J2,5 =1.6 Hz, J2,4
=
=
0.9 Hz, H-2-furyl), 8.17 ppm (s, 1H, H-2); 13C NMR (125.7 MHz,
[D6]DMSO): d=60.37 (CH2-5’), 69.86 (dd, JC,F =26.4 and 17.9 Hz, CH-
3’), 82.12 (dd, JC,F =40.2 and 20.1 Hz, CH-1’), 83.80 (d, JC,F =7.5 Hz,
CH-4’), 100.61 (C-4a), 107.59 (C-5), 111.63 (CH-4-furyl), 118.29 (C-3-
furyl), 121.08 (bd, JC,F =2.6 Hz, CH-6), 123.21 (dd, JC,F =261.8 and
255.0 Hz, C-2’), 140.13 (CH-2-furyl), 144.47 (CH-5-furyl), 151.27
(C-7a), 152.52 (CH-2), 157.75 ppm (C-4); 19F NMR (470.3 MHz,
~
(2ꢄd, 2ꢄ1F, Jgem =234.1 Hz); IR (ATR): n=3456, 3355, 3122, 1626,
1578, 1552, 1449, 1225, 1053, 1026 cmꢀ1; MS (ESI) m/z (%): 369
(100) [M+H], 391 (20) [M+Na]; HRMS-ESI [M+H]+ calcd for
C15H15F2N4O3S: 369.08274, found: 369.08272; Anal. calcd for
C15H14F2N4O3S: C 48.91,H 3.83, N 15.21, found: C 48.69, H 3.79, N
15.11.
[D6]DMSO): d=ꢀ118.70 and ꢀ109.93 ppm (2ꢄd, 2ꢄ1F, Jgem
=
~
233.5 Hz); IR (ATR): n=3499, 3390, 3135, 1622, 1561, 1463, 1303,
1241, 1059, 1040 cmꢀ1; MS (ESI) m/z (%): 353 (100) [M+H], 375
(25) [M+Na]; HRMS-ESI [M+H]+ calcd for C15H15F2N4O4: 353.10559,
found: 353.10557; Anal. calcd for C15H14F2N4O4·1.7H2O: C 47.05, H
4.58, N 14.63, found: C 47.23, H 4.41, N 14.43.
4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
phenyl-pyrrolo[2,3-d]pyrimidine (13e): Compound 13e was pre-
pared as described for compound 11 a from compound 10
(140 mg, 0.34 mmol) and phenylboronic acid (62.2 mg, 0.51 mmol).
The crude product was purified by column chromatography on
silica in 3% CH3OH in CHCl3, and by reversed-phase HPFC on C18
(0!100% CH3OH in H2O), to give compound 13e (80 mg, 65%) as
a white solid after recrystallization (H2O/CH3OH 4:1); mp: 114–
116 8C; [a]D25 = +13.2 cm3 gꢀ1 dmꢀ1 (c=0.170, DMSO); 1H NMR
4-Amino-7-[2-deoxy-2,2-difluoro-a-d-erythro-pentofuranosyl]-5-
(thiophen-2-yl)pyrrolo[2,3-d]pyrimidine (13c): Compound 13c
was prepared as described for compound 11 a from compound 10
(141 mg, 0.34 mmol) and thiophene-2-boronic acid (67.5 mg,
(500 MHz, [D6]DMSO): d=3.59 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH
=
0.51 mmol), to give compound 13c (73 mg, 58%) as a white lyo-
6.3 Hz, J5’a,4’ =4.5 Hz, H-5’a), 3.68 (bdm, 1H, Jgem =12.3 Hz, H-5’b),
4.30 (m, 1H, H-4’), 4.46 (m, 1H, H-3’), 5.11 (dd, 1H, JOH,5’a =6.3 Hz,
25
philizate (tBuOH-benzene 2:1); mp: 87–968C; [a]D
=
+58.7 cm3 gꢀ1 dmꢀ1 (c=0.288, DMSO); 1H NMR (500 MHz,
J
OH,5’b =5.2 Hz, OH-5’), 6.24 (bs, 2H, NH2), 6.47 (d, 1H, JOH,3’ =4.7 Hz,
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OH-3’), 6.68 (dd, 1H, J1’,F =9.3 and 7.9 Hz, H-1’), 7.39 (m, 1H, H-p-
Ph), 7.42 (d, 1H, J6,F =2.9 Hz, H-6), 7.47–7.52 (m, 4H, H-o,m-Ph),
8.20 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.36
(1.50 g, 1.84 mmol) and TsOH·H2O (1.31 g, 3.68 mmol) were dis-
solved in DMF (50 mL) and cooled to 08C. Then, 3,4-dihydro-2H-
pyran (4.73 mL, 27.6 mmol) was added and the reaction mixture
was allowed to warm to room temperature and was stirred over-
night. The reaction mixture was diluted by EtOAc (100 mL) and ex-
tracted with saturated NaHCO3 solution and brine. The organic
layer was dried over MgSO4 and volatiles were thoroughly re-
moved in vacuo. The crude product was purified by column chro-
matography (SiO2, hexane/EtOAc 1:1) to afford compound 17
(1.68 g, 81%) as a pale-yellow foam (mixture of four diastereomers
(CH2-5’), 69.94 (dd, JC,F =26.6 and 18.1 Hz, CH-3’), 82.25 (dd, JC,F
=
40.4 and 20.5 Hz, CH-1’), 83.94 (d, JC,F =7.3 Hz, CH-4’), 100.12 (C-4a),
117.56 (C-5), 121.30 (d, JC,F =2.4 Hz, CH-6), 123.33 (dd, JC,F =262.7
and 252.4 Hz, C-2’), 127.40 (CH-p-Ph), 128.67 and 129.27 (CH-o,m-
Ph), 134.14 (C-i-Ph), 151.41 (C-7a), 152.48 (CH-2), 157.62 ppm (C-4);
19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ118.29 and ꢀ109.16 ppm
~
(2ꢄd, 2ꢄ1F, Jgem =234.3 Hz); IR (ATR): n=3464, 3145, 1636, 1571,
1459, 1303, 1211, 1073, 1041 cmꢀ1; MS (ESI) m/z (%): 363 (100)
[M+H], 385 (18) [M+Na]; HRMS-ESI [M+H]+ calcd for
C17H17F2N4O3: 363.12632, found: 363.12631; Anal. calcd for
C17H16F2N4O3·0.7H2O: C 54.46, H 4.68, N 14.94, found: C 54.28, H
4.41, N 14.70.
1:1:1:1); H NMR (500 MHz, [D6]acetone): d=1.45–1.95 (m, 48H, H-
1
3,4,5-THP), 1.71, 1.75, 1.76 and 1.77 (4s, 4ꢄ3H, CH3), 3.42–3.56 (m,
8H, H-6a-THP), 3.69–3.79 (m, 4H, H-5’a), 3.78–3.96 (m, 8H, H-6b-
THP), 3.97–4.08 (m, 4H, H-5’b), 4.16–4.23 (m, 4H, H-4’), 4.55 (bdd,
1H, J3’,4’ =5.9 Hz, J3’,2’ =4.8 Hz, H-3’), 4.60 (m, 1H, H-3’), 4.63 (bdd,
1H, J3’,4’ =5.7 Hz, J3’,2’ =4.5 Hz, H-3’), 4.69 (bdd, 1H, J3’,4’ =5.9 Hz,
4-Amino-7-[2-O-acetyl-3,5-O-(tetraisopropyldisiloxan-1,3-diyl)-b-
d-arabinofuranosyl]-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (15): To
mixture of arabinoside 14 (2.80 g, 4.41 mmol), Et3N (1.5 mL,
10.6 mmol) and DMAP (54 mg, 0.44 mmol) in anhydrous CH3CN
(150 mL), acetic anhydride (1.0 mL, 10.6 mmol) was added. The re-
action mixture was stirred at room temperature for 1.5 h. Then the
reaction mixture was diluted with EtOAc (100 mL) and extracted
with saturated aqueous solution of NaHCO3 and H2O. The organic
layer was dried over MgSO4 and evaporated under reduced pres-
sure. Acetate 15 (2.98 g, 100%) was obtained as a colorless oil;
1H NMR (500 MHz, CDCl3): d=0.95–1.25 (m, 28H, (CH3)2CH), 1.69 (s,
3H, CH3CO), 3.88 (dt, 1H, J4’,3’ =8.6 Hz, J4’,5’a =J4’,5’b =2.8 Hz, H-4’),
4.05 (dd, 1H, Jgem =13.1 Hz, J5’a,4’ =3.0 Hz, H-5’a), 4.16 (dd, 1H,
J3’,2’ =4.7 Hz, H-3’), 4.72–4.77 (m, 4H, H-2-THP), 4.79–4.84 and 4.87–
4.91 (2 m, 2ꢄ2H, H-2-THP), 5.44 (dd, 1H, J2’,1’ =5.3 Hz, J2’,3’ =4.4 Hz,
H-2’), 5.46 (dd, 1H, J2’,1’ =5.5 Hz, J2’,3’ =4.6 Hz, H-2’), 5.58 (dd, 1H,
J
2’,1’ =5.5 Hz, J2’,3’ =4.8 Hz, H-2’), 5.62 (t, 1H, J2’,1’ =J2’,3’ =5.7 Hz, H-2’),
6.30 (m, 8H, NH2), 6.68–6.70 (m, 2H, H-1’), 6.70 and 6.71 (2ꢄd, 2H,
2 J1’,2’ =5.5 Hz, H-1’), 7.62, 7.64, 7.65 and 7.67 (4 s, 4H, H-6), 8.10,
8.111, 8.113 and 8.115 ppm (4 s, 4H, H-2); 13C NMR (125.7 MHz,
[D6]acetone): d=19.76, 19.81, 19.89, 19.90, 20.07, 20.10, 20.12 and
20.29 (CH2-4-THP), 20.27, 20.34, 20.35 (CH3), 26.02, 26.08, 26.10,
26.18, 26.21, 26.22 and 26.28 (CH2-5-THP), 31.13, 31.17, 31.23,
31.28, 31.31, 31.34, 31.37 and 31.40 (CH2-3-THP), 50.60, 50.62 and
50.68 (C-5), 62.00, 62.08, 62.35, 62.66, 62.72, 62.75 and 62.76 (CH2-
6-THP), 66.56, 66.64, 66.76 and 66.80 (CH2-5’), 76.84, 76.91, 77.08
and 77.35 (CH-2’), 78.1, 78.05, 79.01 and 79.47 (CH-3’), 80.85, 81.04,
81.36 and 81.41 (CH-4’), 82.72, 82.74, 82.90 and 82.99 (CH-1’),
98.48, 98.52, 99.27, 99.30, 99.31, 99.37, 99.46 and 99.48 (CH-2-THP),
104.12, 104.15, 104.18 and 104.19 (C-4a), 128.61, 128.68, 128.78
and 128.82 (CH-6), 151.38, 151.41 and 151.44 (C-7a), 153.27, 153.28
and 153.29 (CH-2), 158.18 and 158.24 (C-4), 169.24, 169.26 and
169.45 ppm (CO); MS (ESI) m/z (%): 603 (15) [M+H], 625 (100)
[M+Na]; HRMS-ESI [M+H]+ calcd for C23H32O7N4I: 603.13102,
found: 603.13118.
J
gem =13.1 Hz, J5’b,4’ =2.6 Hz, H-5’b), 4.69 (t, 1H, J3’,4’ =J3’,2’ =8.5 Hz,
H-3’), 5.58 (dd, 1H, J2’,3’ =8.4 Hz, J2’,1’ =6.5 Hz, H-2’), 6.07 (bs, 2H,
NH2), 6.59 (d, 1H, J1’,2’ =6.5 Hz, H-1’), 7.42 (s, 1H, H-6), 8.22 ppm (s,
1H, H-2); 13C NMR (125.7 MHz, CDCl3): d=12.34, 12.88, 12.98 and
13.41 ((CH3)2CH), 16.73, 16.76, 16.88, 16.94, 17.30, 17.48, 17.55 and
17.71 ((CH3)2CH), 20.13 (CH3CO), 50.66 (C-5), 60.53 (CH2-5’), 70.94
(CH-3’), 76.42 (CH-2’), 80.27 (CH-4’), 80.90 (CH-1’), 103.77 (C-4a),
127.70 (CH-6), 149.90 (C-7a), 150.49 (CH-2), 156.00 (C-4),
169.51 ppm (CO); MS (ESI) m/z (%): 677 (50) [M+H], 699 (100)
[M+Na]; HRMS-ESI [M+H]+ calcd for C25H42O6N4ISi2: 677.16821,
found: 677.16796.
4-Amino-7-[3,5-di-O-(tetrahydropyran-2-yl)-b-d-arabinofurano-
syl]-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (18): Acetate 17 (1.60 g,
2.66 mmol) was dissolved in anhydrous CH3OH (30 mL) and metha-
nolic solution of sodium methoxide (1m, 2.7 mL, 2.7 mmol) was
added. The reaction mixture was stirred at room temperature over-
night and then it was neutralized using aqueous HCl (1m) and
evaporated under reduced pressure. The residue was chromato-
graphed on silica (2% CH3OH in CHCl3) to give compound 18
4-Amino-7-[2-O-acetyl-b-d-arabinofuranosyl]-5-iodo-7H-pyrrolo-
[2,3-d]pyrimidine (16): Silylated acetate 15 (2.98 g, 4.40 mmol)
was dissolved in CH3CN (150 mL) and Et3N·3HF (1.44 mL,
8.81 mmol) was added. The reaction mixture was stirred overnight
at room temperature The crude product was purified by column
chromatography (SiO2, 2% CH3OH in CH2Cl2) to afford acetate 16
(1.56 g, 82%) as a white crystalline solid; 1H NMR (500 MHz,
[D6]DMSO): d=1.68 (s, 3H, CH3CO), 3.63 (ddd, 1H, Jgem =12.1 Hz,
(1.25 g, 84%) as
a
pale-yellow foam; 1H NMR (500 MHz,
[D6]acetone): d=1.47–1.92 (m, 48H, H-3,4,5-THP), 3.47–3.57 (m, 8H,
H-6a-THP), 3.67–3.76 (m, 4H, H-5’a), 3.81–3.99 (m, 8H, H-6b-THP),
3.96–4.07 (m, 4H, H-5’b), 4.12–4.17 (m, 4H, H-4’), 4.38–4.54, 4.60–
5.00, (2 m, 20H, H-2’,3’,2-THP,OH), 6.26 (m, 8H, NH2), 6.53 (d, 1H,
J
J
J
J
5’a,OH =5.8 Hz, J5’a,4’ =4.8 Hz, H-5’a), 3.71 (ddd, 1H, Jgem =12.1 Hz,
5’b,OH =5.5 Hz, J5’b,4’ =3.6 Hz, H-5’b), 3.80 (ddd, 1H, J4’,3’ =6.3 Hz,
4’,5’a =4.8 Hz, J4’,5’b =3.6 Hz, H-4’), 4.30 (dt, 1H, J3’,4’ =6.3 Hz, J3’,OH
=
3’,2’ =5.4 Hz, H-3’), 5.07 (t, 1H, JOH,5’a =JOH,5’b =5.6 Hz, OH-5’), 5.20 (t,
J
1’,2’ =4.7 Hz, H-1’), 6.54 (d, 1H, J1’,2’ =5.0 Hz, H-1’), 6.58 (d, 1H, J1’,2’ =
1H, J2’,1’ =J2’,3’ =5.5 Hz, H-2’), 5.77 (d, 1H, JOH,3’ =5.3 Hz, OH-3’), 6.54
(d, 1H, J1’,2’ =5.6 Hz, H-1’), 6.66 (bs, 2H, NH2), 7.55 (s, 1H, H-6),
8.08 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=20.17
(CH3), 51.69 (C-5), 60.29 (CH2-5’), 71.82 (CH-3’), 77.69 (CH-2’), 81.25
(CH-1’), 82.89 (CH-4’), 102.79 (C-4a), 127.91 (CH-6), 149.95 (C-7a),
152.19 (CH-2), 157.27 (C-4), 169.08 ppm (CO); MS (ESI) m/z (%): 435
(100) [M+H], 457 (73) [M+Na]; HRMS-ESI [M+H]+ calcd for
C13H16O5N4I: 435.01599, found: 435.01598.
4.7 Hz, H-1’), 6.60 (d, 1H, J1’,2’ =5.0 Hz, H-1’), 7.65, 7.66, 7.677 and
7.683 (4 s, 4H, H-6), 8.106, 8.109, 8.114 and 8.12 ppm (4 s, 4H, H-2);
13C NMR (125.7 MHz, [D6]acetone): d=19.99, 20.03, 20.04, 20.07,
20.08, 20.18 and 20.30 (CH2-4-THP), 26.05, 26.07, 26.16, 26.17,
26.18, 26.20 and 26.27 (CH2-5-THP), 31.14, 31.15, 31.21, 31.26,
31.42, 31.50 and 31.58 (CH2-3-THP), 49.74, 49.76, 49.80 and 49.81
(C-5), 61.95, 62.08, 62.14, 62.27, 62.75, 62.81, 63.37 and 63.41 (CH2-
6-THP), 67.27, 67.37, 67.39 and 67.57 (CH2-5’), 76.01, 76.15, 76.23,
76.28, 80.91, 80.99, 81.53, 81.75, 81.84, 81.85, 82.77 and 83.05 (CH-
2’,3’,4’), 84.95, 84.97, 85.10 and 85.26 (CH-1’), 98.63, 98.68, 99.21,
99.32, 99.43, 99.44, 99.48 and 99.67 (CH-2-THP), 104.27 and 104.32
4-Amino-7-[2-O-acetyl-3,5-di-O-(tetrahydropyran-2-yl)-b-d-arabi-
nofuranosyl]-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (17): Acetate 16
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(C-4a), 129.77, 129.82, 129.85 and 129.87 (CH-6), 151.40, 151.41,
151.45 and 151.48 (C-7a), 152.96, 152.97, 152.98 and 153.01 (CH-2),
158.12, 158.15, 158.16 and 158.18 ppm (C-4); MS (ESI) m/z (%): 561
(11) [M+H], 583 (100) [M+Na]; HRMS-ESI [M+H]+ calcd for
C21H30O6N4I: 561.12045, found: 561.12049.
335.11502; Anal. calcd for C15H15O4N4F·2H2O: C 48.65, H 5.17, N
15.13, found: C 48.42, H 4.81, N 14.84.
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-(furan-3-yl)-
7H-pyrrolo[2,3-d]pyrimidine (21b): Nucleoside 21b was prepared
as described for compound 11 a. Nucleoside 20 (120 mg,
0.30 mmol) and furan-3-boronic acid (43 mg, 0.38 mmol) were
used. Nucleoside 21b (65 mg, 64%) was obtained as an off-white
cotton after recrystallization (H2O/CH3OH 4:1); mp: 98–1008C;
[a]D25 =ꢀ39.2 cm3 gꢀ1 dmꢀ1 (c=0.194, DMSO); 1H NMR (500 MHz,
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-iodo-7H-
pyrrolo[2,3-d]pyrimidine (20): Nucleoside 18 (650 mg, 1.16 mmol)
was dissolved in anhydrous CH2Cl2 (16 mL). Pyridine (700 mL,
8.70 mmol) was added and the solution was cooled to 08C. Then
DAST (766 mL, 5.80 mmol) was added. The reaction mixture was al-
lowed to warm to room temperature and was stirred overnight.
The reaction was neutralized with aqueous NaHCO3 (saturated), di-
luted with chloroform (30 mL) and extracted with H2O. The organic
phase was dried over MgSO4 and evaporated under reduced pres-
sure. The crude product 19 was chromatographed on silica
(hexane/EtOAc 2:1) and directly dissolved in 90% aqueous TFA
(5 mL). The solution was stirred for 2 h and then it was repeatedly
co-evaporated with CH3OH. The final product was purified by
column chromatography (silica, 2.5% CH3OH in CHCl3) to afford flu-
oronucleoside 20 (163 mg, 36% over two steps) as a white crystal-
[D6]DMSO): d=3.58 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH =5.8 Hz, J5’a,4’
4.0 Hz, H-5’a), 3.71 (ddd, 1H, Jgem =12.3 Hz, J5’b,OH =5.3 Hz, J5’b,4’
3.0 Hz, H-5’b), 3.94 (m, 1H, H-4’), 4.39 (dtd, 1H, J3’,F =15.5 Hz, J3’,4’
=
=
=
J
3’,OH =5.9 Hz, J3’,2’ =4.7 Hz, H-3’), 5.18 (t, 1H, JOH,5’a =JOH,5’b =5.5 Hz,
OH-5’), 5.28 (ddd, 1H, J2’,F =53.1 Hz, J2’,3’ =4.6 Hz, J2’,1’ =3.7 Hz, H-2’),
5.68 (d, 1H, JOH,3’ =5.9 Hz, OH-3’), 6.32 (bs, 2H, NH2), 6.39 (dd, 1H,
J
1’,F =16.6 Hz, J1’,2’ =3.7 Hz, H-1’), 6.70 (dd, 1H, J4,5 =1.8 Hz, J4,2 =
0.9 Hz, H-4-furyl), 7.52 (s, 1H, H-6), 7.81 (t, 1H, J5,4 =J5,2 =1.7 Hz, H-
5-furyl), 7.74 (dd, 1H, J2,5 =1.6 Hz, J2,4 =0.9 Hz, H-2-furyl), 8.15 ppm
(s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.75 (CH2-5’),
68.66 (d, JC,F =15.7 Hz, CH-3’), 83.97 (d, JC,F =1.9 Hz, CH-4’), 85.16 (d,
1
line solid after recrystallization (H2O/CH3OH 3:1); H NMR (500 MHz,
JC,F =32.3 Hz, CH-1’), 93.75 (d, JC,F =187.5 Hz, C-2’), 101.11 (C-4a),
[D6]DMSO): d=3.57 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH =5.7 Hz, J5’a,4’
3.7 Hz, H-5’a), 3.71 (ddd, 1H, Jgem =12.2 Hz, J5’b,OH =5.2 Hz, J5’b,4’
2.9 Hz, H-5’b), 3.92 (m, 1H, H-4’), 4.35 (dtd, 1H, J3’,F =16.1 Hz, J3’,4’
=
=
=
106.94 (C-5), 111.66 (CH-4-furyl), 118.54 (C-3-furyl), 120.72 (CH-6),
139.93 (CH-2-furyl), 144.39 (CH-5-furyl), 150.53 (C-7a), 152.23 (CH-2),
157.71 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
~
J
3’,OH =6.0 Hz, J3’,2’ =4.7 Hz, H-3’), 5.19 (t, 1H, JOH,5’a =JOH,5’b =5.4 Hz,
OH-5’), 5.22 (ddd, 1H, J2’,F =53.1 Hz, J2’,3’ =4.7 Hz, J2’,1’ =3.5 Hz, H-2’),
5.67 (d, 1H, JOH,3’ =6.0 Hz, OH-3’), 6.32 (dd, 1H, J1’,F =16.5 Hz, J1’,2’
ꢀ200.82 ppm (s, 1F, F-2’); IR (ATR): n=3367, 3139, 1626, 1566,
1470, 1454, 1096, 1022 cmꢀ1; MS (ESI) m/z (%): 335 (100) [M+H],
357 (27) [M+Na]; HRMS-ESI [M+H]+ calcd for C15H16O4N4F:
335.11501, found: 335.11505; Anal. calcd for C15H15O4N4F·1.5H2O: C
49.86, H 5.02, N 15.51, found: C 50.22, H 4.65, N 15.35.
=
3.5 Hz, H-1’), 6.73 (bs, 2H, NH2), 7.69 (s, 1H, H-6), 8.12 ppm (s, 1H,
H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=52.67 (C-5), 60.52 (CH2-
5’), 68.45 (d, JC,F =15.8 Hz, CH-3’), 83.99 (d, JC,F =2.0 Hz, CH-4’),
85.24 (d, JC,F =32.6 Hz, CH-1’), 93.78 (d, JC,F =187.3 Hz, C-2’), 103.41
(C-4a), 126.92 (CH-6), 149.89 (C-7a), 152.39 (CH-2), 157.45 ppm (C-
4); 19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ200.75 ppm (s, 1F, F-2’);
MS (ESI) m/z (%): 395 (100) [M+H], 417 (70) [M+Na]; HRMS-ESI
[M+H]+ calcd for C11H13O3N4FI: 395.00109, found: 395.00114.
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-(thiophen-2-
yl)-7H-pyrrolo[2,3-d]pyrimidine (21c): Nucleoside 21c was pre-
pared as described for compound 11 a. Nucleoside 20 (120 mg,
0.30 mmol) and thiophene-2-boronic acid (49 mg, 0.38 mmol) were
used. Nucleoside 21c (57 mg, 53%) was obtained as a white lyo-
25
philizate (tBuOH-benzene 3:1); mp: 96–1018C; [a]D
=
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-(furan-2-yl)-
7H-pyrrolo[2,3-d]pyrimidine (21a): Nucleoside 21a was prepared
as described for compound 11 a from nucleoside 20 (120 mg,
0.30 mmol) and furan-2-boronic acid (43 mg, 0.38 mmol). The
crude product was purified by reversed-phase HPFC on C18 column
(0!100% CH3OH in H2O). Nucleoside 21a (54 mg, 53%) was ob-
tained as a white crystalline solid after recrystallization (H2O/CH3OH
4:1); mp: 103–1058C; [a]D25 =ꢀ45.0 cm3 gꢀ1 dmꢀ1 (c=0.251,
ꢀ40.2 cm3 gꢀ1 dmꢀ1 (c=0.293, DMSO); 1H NMR (500 MHz,
[D6]DMSO): d=3.58 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH =5.5 Hz, J5’a,4’
3.9 Hz, H-5’a), 3.73 (ddd, 1H, Jgem =12.3 Hz, J5’b,OH =5.0 Hz, J5’b,4’
2.9 Hz, H-5’b), 3.95 (m, 1H, H-4’), 4.40 (bdt, 1H, J3’,F =16.1 Hz, J3’,2’
=
=
=
J
3’,4’ =5.3 Hz, H-3’), 5.21 (bt, 1H, JOH,5’a =JOH,5’b =5.3 Hz, OH-5’), 5.30
(ddd, 1H, J2’,F =53.1 Hz, J2’,3’ =4.7 Hz, J2’,1’ =3.5 Hz, H-2’), 5.70 (bs,
1H, OH-3’), 6.37 (bs, 2H, NH2), 6.40 (dd, 1H, J1’,F =16.5 Hz, J1’,2’
3.5 Hz, H-1’), 7.16 (dd, 1H, J3,4 =3.5 Hz, J3,5 =1.3 Hz, H-3-thienyl),
7.18 (dd, 1H, J4,5 =5.1 Hz, J4,3 =3.5 Hz, H-4-thienyl), 7.58 (dd, 1H,
5,4 =5.1 Hz, J5,3 =1.3 Hz, H-5-thienyl), 7.66 (s, 1H, H-6), 8.18 ppm (s,
1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.54 (CH2-5’), 68.51
(d, JC,F =15.7 Hz, CH-3’), 83.99 (d, JC,F =2.0 Hz, CH-4’), 85.33 (d, JC,F
32.3 Hz, CH-1’), 93.83 (d, JC,F =187.7 Hz, C-2’), 100.76 (C-4a), 109.12
(C-5), 121.82 (CH-6), 126.15 (CH-5-thienyl), 126.69 (CH-3-thienyl),
128.48 (CH-4-thienyl), 135.42 (C-2-thienyl), 150.39 (C-7a), 152.48
(CH-2), 157.52 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
=
DMSO); 1H NMR (500 MHz, [D6]DMSO): d=3.60 (ddd, 1H, Jgem
12.3 Hz, J5’a,OH =5.7 Hz, J5’a,4’ =3.9 Hz, H-5’a), 3.75 (ddd, 1H, Jgem
=
=
J
12.3 Hz, J5’b,OH =5.3 Hz, J5’b,4’ =2.9 Hz, H-5’b), 3.96 (m, 1H, H-4’), 4.40
(dtd, 1H, J3’,F =16.6 Hz, J3’,4’ =J3’,OH =6.0 Hz, J3’,2’ =4.6 Hz, H-3’), 5.22
(t, 1H, JOH,5’a =JOH,5’b =5.5 Hz, OH-5’), 5.27 (ddd, 1H, J2’,F =53.1 Hz,
=
J
2’,3’ =4.6 Hz, J2’,1’ =3.4 Hz, H-2’), 5.70 (d, 1H, JOH,3’ =6.0 Hz, OH-3’),
6.39 (dd, 1H, J1’,F =16.4 Hz, J1’,2’ =3.4 Hz, H-1’), 6.62 (dd, 1H, J4,3
=
3.3 Hz, J4,5 =1.9 Hz, H-4-furyl), 6.66 (dd, 1H, J3,4 =3.3 Hz, J3,5 =0.8 Hz,
H-3-furyl), 6.95 (bs, 2H, NH2), 7.79 (dd, 1H, J5,4 =1.9 Hz, J5,3 =0.8 Hz,
H-5-furyl), 7.86 (s, 1H, H-6), 8.16 ppm (s, 1H, H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=60.62 (CH2-5’), 68.54 (d, JC,F =15.8 Hz,
CH-3’), 83.95 (d, JC,F =1.8 Hz, CH-4’), 85.34 (d, JC,F =32.4 Hz, CH-1’),
93.83 (d, JC,F =187.3 Hz, C-2’), 99.45 (C-4a), 105.67 (CH-3-furyl),
106.79 (C-5), 112.10 (CH-4-furyl), 120.20 (CH-6), 142.33 (CH-5-furyl),
148.55 (C-2-furyl), 150.63 (C-7a), 152.57 (CH-2), 157.51 ppm (C-4);
19F NMR (470.3 MHz, [D6]DMSO): d=ꢀ200.38 ppm (s, 1F, F-2’); IR
~
ꢀ200.58 ppm (s, 1F, F-2’); IR (ATR): n=3340, 3194, 1622, 1588,
1551, 1465, 1290, 1190, 1096, 1063; MS (ESI) m/z (%): 351 (100)
[M+H], 373 (40) [M+Na]; HRMS-ESI [M+H]+ calcd for
C15H16O3N4FS: 351.09217, found: 351.09234; Anal. calcd for
C15H16O3N4FS: C 51.42, H 4.32, N 15.99, found: C 51.02, H 4.68, N
15.73.
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-(thiophen-3-
yl)-7H-pyrrolo[2,3-d]pyrimidine (21d): Nucleoside 21d was pre-
pared as described for compound 11 a. Nucleoside 20 (120 mg,
0.30 mmol) and thiophene-3-boronic acid (49 mg, 0.38 mmol) were
~
(ATR): n=3455, 3128, 1635, 1561, 1482, 1460, 1297, 1234, 1109,
1067, 1043, 1017; MS (ESI) m/z (%): 335 (98) [M+H], 357 (100) [M+
Na]; HRMS-ESI [M+H]+ calcd for C15H16O4N4F: 335.11501, found:
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 832 – 846 843

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used. Nucleoside 21d (65 mg, 61%) was obtained as a white crys-
J
J
3’,F =16.8 Hz, J3’,OH =J3’,4’ =6.2 Hz, J3’,2’ =4.6 Hz, H-3’), 5.23 (t, 1H,
OH,5’a =JOH,5’b =5.4 Hz, OH-5’), 5.24 (ddd, 1H, J2’,F =53.0 Hz, J2’,3’ =
25
talline solid (H2O/CH3OH 4:1); mp: 106–1088C; [a]D
=
ꢀ34.7 cm3 gꢀ1 dmꢀ1 (c=0.285, DMSO); 1H NMR (500 MHz,
4.6 Hz, J2’,1’ =3.2 Hz, H-2’), 5.70 (d, 1H, JOH,3’ =6.0 Hz, OH-3’), 6.32
(dd, 1H, 1’,F =16.5 Hz, 1’,2’ =3.3 Hz, H-1’), 7.84 (s, 1H, H-6),
[D6]DMSO): d=3.58 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH =5.8 Hz, J5’a,4’
4.0 Hz, H-5’a), 3.72 (ddd, 1H, Jgem =12.3 Hz, J5’b,OH =5.2 Hz, J5’b,4’
=
=
J
J
8.15 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.45
(CH2-5’), 68.42 (d, JC,F =15.9 Hz, CH-3’), 77.24 (CꢁCH), 83.54 (Cꢁ
CH), 84.03 (d, JC,F =1.8 Hz, CH-4’), 85.54 (d, JC,F =32.6 Hz, CH-1’),
93.90 (d, JC,F =187.5 Hz, C-2’), 94.62 (C-5), 102.52 (C-4a), 127.21 (CH-
6), 149.30 (C-7a), 153.27 (CH-2), 157.77 ppm (C-4); 19F NMR
2.9 Hz, H-5’b), 3.95 (m, 1H, H-4’), 4.40 (bdtd, 1H, J3’,F =15.6 Hz,
J
3’,OH =J3’,4’ =5.8 Hz, J3’,2’ =4.5 Hz, H-3’), 5.19 (t, 1H, JOH,5’a =JOH,5’b
=
5.5 Hz, OH-5’), 5.30 (ddd, 1H, J2’,F =53.0 Hz, J2’,3’ =4.5 Hz, J2’,1’
=
3.7 Hz, H-2’), 5.69 (d, 1H, JOH,3’ =5.8 Hz, OH-3’), 6.26 (bs, 2H, NH2),
~
6.40 (dd, 1H, J1’,F =16.7 Hz, J1’,2’ =3.7 Hz, H-1’), 7.27 (dd, 1H, J4,5
4.9 Hz, J4,2 =1.3 Hz, H-4-thienyl), 7.53 (dd, 1H, J2,5 =2.9 Hz, J2,4
=
=
(470.3 MHz, [D6]DMSO): d=ꢀ200.42 ppm (s, 1F, F-2’); IR (KBr): n=
3508, 3467, 3368, 3167, 1641, 1599, 1573, 1530, 1448, 1367, 1314,
1199, 1059, 1033, 1017; MS (ESI) m/z (%): 293 (100) [M+H], 315
(95) [M+Na]; HRMS-ESI [M+H]+ calcd for C13H14O3N4F: 293.10445,
found: 293.10459; Anal. calcd for C13H13O3N4F·0.5 CH3OH: C 52.60,
H 4.90, N 18.17, found: C 52.47, H 4.67, N 17.93.
1.3 Hz, H-2-thienyl), 7.57 (s, 1H, H-6), 7.72 (dd, 1H, J5,4 =4.9 Hz,
5,2 =2.9 Hz, H-5-thienyl), 8.16 ppm (s, 1H, H-2); 13C NMR
J
(125.7 MHz, [D6]DMSO): d=60.73 (CH2-5’), 68.66 (d, JC,F =15.7 Hz,
CH-3’), 83.99 (d, JC,F =1.9 Hz, CH-4’), 85.21 (d, JC,F =32.3 Hz, CH-1’),
93.81 (d, JC,F =187.3 Hz, C-2’), 100.92 (C-4a), 111.66 (C-5), 120.84
(CH-6), 122.41 (CH-2-thienyl), 127.64 (CH-5-thienyl), 128.63 (CH-4-
thienyl), 134.67 (C-3-thienyl), 150.40 (C-7a), 152.22 (CH-2),
157.65 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(thiophene-2-yl)pyrrolo[2,3-d]pyrimidine-5’-O-triphosphate
sodium salt (11c-TP): Compound 11 c (30 mg, 0.081 mmol) was
dissolved in trimethyl phosphate (350 mL) in argon-purged vial and
the suspension was cooled to 08C. Then POCl3 (9.5 mL,
0.102 mmol) was added. The reaction mixture was stirred at 08C
for 2.5 h and then ice-cold solution of (NHBu3)2H2P2O7 (234.3 mg,
0.427 mmol) and tri(n-butyl)amine (92 mL, 0.387 mmol) in anhy-
drous DMF (1 mL) was added. The reaction mixture was stirred at
ꢀ58C for 2.5 h. Aqueous solution of TEAB (2m, 3 mL, 6 mmol) was
added and the mixture was evaporated under reduced pressure.
The residue was co-evaporated several times with H2O. The prod-
uct was purified on DEAE Sephadex column (0–1.2m TEAB gradi-
ent), co-evaporated several times with H2O and converted into
a sodium salt form (Dowex 50WX8 in Na+ cycle). Triphosphate
11c-TP was obtained as a white lyophilizate (H2O; 19 mg, 36%);
1H NMR (500 MHz, D2O): d=4.27 (m, 1H, H-4’), 4.35 (ddd, 1H,
~
ꢀ200.72 ppm (s, 1F, F-2’); IR (ATR): n=3373, 3106, 1620, 1550,
1463, 1300, 1127, 1097, 1041; MS (ESI) m/z (%): 351 (100) [M+H],
373 (75) [M+Na]; HRMS-ESI [M+H]+ calcd for C15H16O3N4FS:
351.09217, found: 351.09214; Anal. calcd for C15H16O3N4FS·2H2O: C
46.63, H 4.96, N 14.50, found: C 47.00, H 4.68, N 14.42.
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-phenyl-7H-
pyrrolo[2,3-d]pyrimidine (21e): Nucleoside 21e was prepared as
described for compound 11 a. Nucleoside 20 (150 mg, 0.38 mmol)
and phenylboronic acid (58 mg, 0.47 mmol) were used. Nucleoside
21e (91 mg, 69%) was obtained as a white lyophilizate (tBuOH-
benzene 2:1); mp: 93–958C; [a]D25 =ꢀ40.8 cm3 gꢀ1 dmꢀ1 (c=0.446,
DMSO); 1H NMR (500 MHz, [D6]DMSO): d=3.58 (dd, 1H, Jgem
12.3 Hz, J5’a,4’ =3.9 Hz, H-5’a), 3.72 (dd, 1H, Jgem =12.3 Hz, J5’b,4’
2.9 Hz, H-5’b), 3.95 (m, 1H, H-4’), 4.41 (ddd, 1H, J3’,F =15.9 Hz, J3’,4’
=
=
=
J
gem =12.0 Hz, J5’a,P =6.5 Hz, J5’a,4’ =3.8 Hz, H-5’a), 4.39 (m, 1H, H-
5’b), 4.66 (ddd, 1H, J3’,F =12.6 and 11.6 Hz, J3’,4’ =8.3 Hz, H-3’), 6.45
(dd, 1H, J1’,F =10.9 and 5.1 Hz, H-1’), 7.12 (dd, H, J3,4 =3.5 Hz, J3,5
6.0 Hz, J3’,2’ =4.7 Hz, H-3’), 5.32 (ddd, 1H, J2’,F =53.1 Hz, J2’,3’ =4.7 Hz,
2’,1’ =3.6 Hz, H-2’), 6.42 (dd, 1H, J1’,F =16.7 Hz, J1’,2’ =3.6 Hz, H-1’),
J
=
7.38 (m, 1H, H-p-Ph), 7.46–7.52 (m, 4H, H-o,m-Ph), 7.58 (s, 1H, H-6),
8.18 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, [D6]DMSO): d=60.70
(CH2-5’), 68.65 (d, JC,F =15.8 Hz, CH-3’), 83.99 (d, JC,F =1.9 Hz, CH-4’),
85.27 (d, JC,F =32.4 Hz, CH-1’), 93.88 (d, JC,F =187.2 Hz, C-2’), 100.61
(C-4a), 116.99 (C-5), 120.94 (CH-6), 127.24 (CH-p-Ph), 128.65 and
129.22 (CH-o,m-Ph), 134.46 (C-i-Ph), 150.68 (C-7a), 152.20 (CH-2),
157.58 ppm (C-4); 19F NMR (470.3 MHz, [D6]DMSO): d=
1.2 Hz, H-3-thienyl), 7.17 (dd, 1H, J4,5 =5.2 Hz, J4,3 =3.5 Hz, H-4-
thienyl), 7.49 (dd, 1H, J5,4 =5.2 Hz, J5,3 =1.2 Hz, H-5-thienyl), 7.50
(bs, 1H, H-6), 8.16 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, D2O): d=
61.18 (d, JC,P =5.1 Hz, CH2-5’), 69.61 (dd, JC,F =27.5 and 17.8 Hz, CH-
3’), 79.51 (t, JC,F =JC,P =8.9 Hz, CH-4’), 83.44 (dd, JC,F =40.8 and
23.6 Hz, CH-1’), 101.46 (C-4a), 111.87 (C-5), 122.45 (CH-6), 122.76
(dd, JC,F =261.8 and 255.4 Hz, C-2’), 127.22 (CH-5-thienyl), 128.01
(CH-3-thienyl), 128.87 (CH-4-thienyl), 134.31 (C-2-thienyl), 149.99
(C-7a), 151.39 (CH-2), 156.97 ppm (C-4); 19F NMR (470.3 MHz, D2O):
~
ꢀ200.56 ppm (s, 1F, F-2’); IR (KBr): n=3351, 3199, 1623, 1584, 1540,
1466, 1297, 1104, 1065; MS (ESI) m/z (%): 345 (100) [M+H], 367
(30) [M+Na]; HRMS-ESI [M+H]+ calcd for C17H18O3N4F: 345.13575,
found: 345.13569; Anal. calcd for C17H17O3N4F: C 59.30, H 4.98, N
16.27, found: C 59.00, H 4.98, N 16.21.
d=ꢀ114.64 and ꢀ113.23 ppm (2ꢄd, 2ꢄ1F,
Jgem =238.2 Hz);
31P NMR (202.3 MHz, D2O): d=ꢀ21.54 (t, 1P, Jb,a =Jb,g =19.0 Hz, Pb),
ꢀ10.25 (d, 1P, Ja,b =19.2 Hz, Pa), ꢀ8.38 ppm (d, 1P, Jg,b =18.7 Hz,
Pg); MS (ESIꢀ) m/z (%): 650 (10) [M+2Na], 628 (5) [M+Na+H],
549 (45) [MꢀPO3 +Na+H], 527 (100) [MꢀPO3 +2H], 447 (35)
4-Amino-7-[2-deoxy-2-fluoro-b-d-ribofuranosyl]-5-ethynyl-7H-
pyrrolo[2,3-d]pyrimidine (21h): An argon-purged mixture of deriv-
ative 20 (130 mg, 0.33 mmol), PdCl2(PPh3)2 (12 mg, 16 mmol), CuI
(6.3 mg, 33 mmol), trimethylsilylacetylene (470 mL, 3.3 mmol) and
triethylamine (150 mL) was stirred in DMF (600 mL) at room temper-
ature overnight. Volatiles were removed under reduced pressure
and the crude product was purified by column chromatography
on silica in 2.5% CH3OH in CHCl3. Then, the product was purified
by reversed-phase HPFC on C18 (0!100% CH3OH in H2O) to give
[Mꢀ2PO3 +H];
HRMS-ESI
[M+Na+H]ꢀ
calcd
for
C15H15F2N4NaO12P3S: 628.94913, found: 628.94963.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
(benzofuran-2-yl)pyrrolo[2,3-d]pyrimidine-5’-O-triphosphate
sodium salt (11g-TP): Compound 11g-TP was prepared as de-
scribed for compound 11c-TP from compound 11 g (28 mg,
0.070 mmol), with POCl3 (8.17 mL, 0.087 mmol), (NHBu3)2H2P2O7
ethynyl derivative 21h (73 mg, 76%) as a beige crystalline solid
(200.5 mg,
0.365 mmol),
and
tri(n-butyl)amine
(78.8 mL,
25
after recrystallization (CH3OH/H2O 1:4); mp: 243–2488C; [a]D
=
0.331 mmol) to give the triphosphate 11g-TP (6.2 mg, 13%) as
ꢀ42.7 cm3 gꢀ1 dmꢀ1 (c=0.446, DMSO); 1H NMR (500 MHz,
a white lyophlizate (H2O); H NMR (500 MHz, D2O): d=4.24 (m, 1H,
1
[D6]DMSO): d=3.58 (ddd, 1H, Jgem =12.3 Hz, J5’a,OH =5.7 Hz, J5’a,4’
3.7 Hz, H-5’a), 3.73 (ddd, 1H, Jgem =12.3 Hz, J5’b,OH =5.2 Hz, J5’b,4’
=
=
H-4’), 4.38–4.49 (m, 2H, H-5’), 4.76 (m, 1H, H-3’), 6.09 (bdd, 1H,
J
1’,F =10.8 and 4.3 Hz, H-1’), 6.86 (s, 1H, H-3-benzofuryl), 7.15–7.21
2.9 Hz, H-5’b), 3.94 (m, 1H, H-4’), 4.30 (s, 1H, CꢁCH), 4.37 (dtd, 1H,
(m, 2H, H-5,6-benzofuryl), 7.29 (m, 1H, H-7-benzofuryl), 7.43 (m,
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1H, H-4-benzofuryl), 7.71 (s, 1H, H-2), 7.79 ppm (s, 1H, H-6);
13C NMR (125.7 MHz, D2O): d=63.92 (d, JC,P =5.0 Hz, CH2-5’), 69.26
(dd, JC,F =27.6 and 17.5 Hz, CH-3’), 79.62 (t, JC,F =JC,P =8.9 Hz, CH-4’),
83.22 (dd, JC,F =40.7 and 23.7 Hz, CH-1’), 99.71 (C-4a), 102.29 (CH-3-
benzofuryl), 108.48 (C-5), 111.22 (CH-7-benzofuryl), 121.31 (CH-4-
benzofuryl), 121.54 (CH-6), 122.90 (dd, JC,F =261.9 and 255.5 Hz, C-
2’), 123.93 (CH-5-benzofuryl), 124.46 (CH-6-benzofuryl), 128.97
(C-3a-benzofuryl), 149.88 (C-2-benzofuryl), 149.94 (C-7a), 151.52
(CH-2), 153.90 (C-7a-benzofuryl), 157.04 ppm (C-4); 19F NMR
(470.3 MHz, D2O): d=ꢀ114.40 and ꢀ112.86 ppm (2ꢄd, 2ꢄ1F,
KOD XL (2.5 UmLꢀ1, 0.087 mL), or Pwo (1 UmLꢀ1, 2.0 mL)), natural
dNTPs (4 mm, 0.5 mL), functionalized triphosphate 11c-TP, 11g-TP
(4 mm, 1 mL), or 11h-TP (4 mm, 2 mL), 5’-32P-labeled primer (3 mm,
0.72 mL, Prim248short: 3’-GGG TAC GGC GGG TAC-5’), and 31-mer tem-
plate (3 mm, 1.09 mL, TempPrb4basII: 5’-CTA GCA TGA GCT CAG TCC
CAT GCC GCC CAT G-3’) in the buffer (2 mL) supplied by the manu-
facturer with the enzyme (ThermoPol buffer for DeepVent(exoꢀ)
and Vent(exoꢀ), KOD XL buffer, or Pwo buffer). The primer was la-
beled using [g32P]ATP according to standard techniques.[15] Reac-
tion mixtures were incubated at 608C for 20 min (for KOD XL the
incubation time was only 10 min). The reactions were stopped by
the addition of PAGE stop solution (40 mL, 80% v/v formamide,
20 mm EDTA, 0.025% w/v bromophenol blue, 0.025% w/v xylene
cyanol) and heated at 958C for 5 min. Aliquots (4 mL) were subject-
ed to vertical electrophoresis in 12.5% denaturing polyacrylamide
gel containing 1ꢄ TBE buffer (pH 8) and 7m urea at 45 mA for
50 min. The gels were dried (858C 70 min), audioradiographed and
visualized by phosphorimager (Typhoon 9410, Amersham Bio-
sciences).
J
J
gem =238.0 Hz); 31P NMR (202.3 MHz, D2O): d=ꢀ20.60 (t, 1P, Jb,a
=
b,g =19.8 Hz, Pb), ꢀ10.04 (d, 1P, Ja,b =19.3 Hz, Pa), ꢀ5.20 ppm (d, 1P,
Jg,b =20.2 Hz, Pg); MS (ESIꢀ) m/z (%): 685 (20) [M+2Na+H], 663
(15) [M+Na+H], 583 (100) [MꢀPO3 +Na+H], 561 (98) [MꢀPO3 +
2H], 481 (80) [Mꢀ2PO3 +H]; HRMS-ESI [M+Na+H]ꢀ calcd for
C19H15F2N4NaO13P3: 662.98762, found: 662.98747.
4-Amino-7-[2-deoxy-2,2-difluoro-b-d-erythro-pentofuranosyl]-5-
ethynyl-pyrrolo[2,3-d]pyrimidine-5’-O-triphosphate sodium salt
(11h-TP): Compound 11h-TP was prepared as described for com-
pound 11c-TP from compound 11 h (30 mg, 0.097 mmol), with
Primer extension experiments with bacterial DNA polymerases for
MALDI-TOF analysis: The reaction mixture (100 mL) contained Vent-
(exoꢀ) DNA polymerase (2 UmLꢀ1, 1 mL), primer Prim248 short (10 mm,
10 mL), 5’-biotinylated template TempPrb4basII–bio (10 mm, 15 mL), natu-
ral dNTPs (4 mm, 5 mL) and the modified dNTP 11c-TP, 11g-TP or
11h-TP (4 mm, 10 mL) in ThermoPol reaction buffer (10 mL). The re-
action mixture was incubated at 608C for 20 min in a thermal
cycler. The reaction was stopped by cooling to 48C. Streptavidin
magnetic particles (Roche, 500 mL) were washed with binding
buffer TEN100 (10 mm Tris, 1 mm EDTA, 100 mm NaCl, pH 7.5; 3ꢄ
750 mL). The reaction mixture after PEX was diluted with the bind-
ing buffer TEN100 (100 mL), the solution was added to the pre-
washed magnetic beads and incubated for 20 min at 188C and
1200 rpm. After the incubation, the magnetic beads were collected
on a magnet (PureProteome Magnetic Stand, Merck) and the solu-
tion was discarded. The beads were washed successively with
wash buffer TEN1000 (10 mm Tris, 1 mm EDTA, 1m NaCl, pH 7.5; 2ꢄ
750 mL), and water (3ꢄ750 mL). Then water (50 mL) was added and
the sample was denatured for 2 min at 658C and 900 rpm. The
beads were collected on a magnet and the solution was trans-
ferred into a clean vial. The product was analyzed by MALDI-TOF
mass spectrometry.
POCl3
(11.27 mL,
0.121 mmol),
(NHBu3)2H2P2O7
(278.5 mg,
0.508 mmol), and tri(n-butyl)amine (109.4 mL, 0.459 mmol) to give
the triphosphate 11h-TP (6.8 mg, 12%) as a white lyophlizate
(H2O); 1H NMR (500 MHz, D2O): d=3.72 (s, 1H, CHꢁC), 4.28 (m,
1H, H-4’), 4.36 (ddd, 1H, Jgem =12.2 Hz, J5’a,P =6.5 Hz, J5’a,4’ =3.5 Hz,
H-5’a), 4.44 (bdm, 1H, Jgem =12.2 Hz, H-5’b), 4.66 (m, 1H, H-3’), 6.41
(dd, 1H, J1’,F =10.6 and 4.3 Hz, H-1’), 7.71 (d, 1H, J6,1’ =1.1 Hz, H-6),
8.14 ppm (s, 1H, H-2); 13C NMR (125.7 MHz, D2O): d=63.98 (d, JC,P
=
5.4 Hz, CH2-5’), 69.10 (dd, JC,F =26.7 and 18.3 Hz, CH-3’), 75.86
(CHꢁC), 79.70 (bt, JC,F =JC,P =8.6 Hz, CH-4’), 83.54 (dd, JC,F =40.5
and 24.1 Hz, CH-1’), 97.43 (C-5), 100.67 (CHꢁC), 103.20 (C-4a),
122.67 (dd, JC,F =261.7 and 255.4 Hz, C-2’), 128.43 (CH-6), 148.52 (C-
7a), 151.11 (CH-2), 156.29 ppm (C-4); 19F NMR (470.3 MHz, D2O): d=
ꢀ115.13 and ꢀ114.10 ppm (2ꢄd, 2ꢄ1F, Jgem =238.4 Hz); 31P NMR
(202.3 MHz, D2O): d=ꢀ21.75 (t, 1P, Jb,a =Jb,g =19.4 Hz, Pb), ꢀ10.27
(d, 1P, Ja,b =19.4 Hz, Pa), ꢀ9.20 ppm (d, 1P, Jg,b =19.4 Hz, Pg); MS
(ESIꢀ) m/z (%): 571 (15) [M+NaꢀH], 549 (100) [MꢀH]; HRMS-ESI
[MꢀH]ꢀ calcd for C13H14F2N4O12P3: 548.97946, found: 548.97967.
Gemcitabine triphosphate sodium salt (4-TP): Dry POCl3 (12 mL,
0.12 mmol) was added to a solution of gemcitabine hydrochloride
(4, 19 mg, 0.06 mmol) and proton sponge (13 mg, 0.06 mmol) in
dry trimethyl phosphate (1 mL) at 08C under argon atmosphere.
The reaction mixture was stirred at 08C for 24 h. Next, an ice-cold
solution of (NHBu3)2H2P2O7 (173 mg, 0.32 mmol) and tri(n-butyl)a-
mine (100 mL, 0.42 mmol) in anhydrous DMF (1 mL) was added.
The reaction mixture was stirred at 08C for 45 min. The reaction
was quenched with aqueous solution of TEAB (2m, 1 mL) and the
mixture was concentrated under reduced pressure. The residue
was co-evaporated five times with H2O. The product was purified
on DEAE Sephadex column (0–1.2m TEAB gradient). The obtained
triphosphate was converted into the sodium salt using Ionex
(Dowex 50WX8 in Na+ cycle) and freeze-dried from H2O. Com-
pound 4-TP was obtained as white solid (14 mg, 37%). NMR data
are in accord with published values.[14] 1H NMR (500 MHz, D2O):
4.15–4.20 (m, 1H), d=4.34–4.38 (m, 2H), 4.53–4.63 (m, 1H), 6.14
(d, 1H, J=7.7 Hz), 6.25 (t, 1H, J=7.2 Hz), 7.90 ppm (d, 1H, J=
7.6 Hz); 31P NMR (202.3 MHz, D2O): d=ꢀ21.54 (t, 1P, J=19.5 Hz),
ꢀ11.10 (d, 1P, J=19.5 Hz), ꢀ6.21 ppm (d, 1P, J=20.1 Hz).
Primer annealing: The radiolabeled primer (Prim248short: 3’-GGG TAC
GGC GGG TAC-5’) was mixed with the template (TempMonoT: 5’-CTA
GCA TGA GCT CAG ACC CAT GCC GCC CAT G-3’, 1.5-fold excess) in
aqueous Tris·HCl (pH 7.5, 50 mm), DTT (5 mm), MgCl2 (5 mm), so
that the final primer concentration was 1.1 mm. The annealing was
performed in a thermal cycler. The sample was first heated at 958C
for 5 min and then slowly cooled to 258C over 60 min. Thus pre-
pared primed template was stored at ꢀ208C.
Primer extension experiments using human DNA polymerase a: The
reaction mixture (10 mL) contained primed template (0.1 pm
primer, 0.15 pm template), human DNA polymerase a (4 UmLꢀ1, 0.5
U), BSA (0.1 mgmLꢀ1), glycerol (10%), Tris·HCl (pH 7.5, 50 mm), DTT
(5 mm), MgCl2 (5 mm), natural dNTPs (100 mm) and/or modified
dNTP (200 mm). The reactions were carried out at 378C for 60 min
and were stopped by the addition of 20 mL PAGE stop solution
(80% v/v formamide, 20 mm EDTA, 0.025% w/v bromophenol blue,
0.025% w/v xylene cyanol) and heated at 958C for 5 min. Aliquots
(4 mL) were subjected to vertical electrophoresis in 12.5% denatur-
ing polyacrylamide gel containing 1ꢄ TBE buffer (pH 8) and 7m
urea at 45 mA for 50 min. The gels were dried (858C 70 min),
Primer extension experiments with bacterial DNA polymerases: The
reaction mixture for PEX (20 mL) contained DNA polymerase (Deep
, , 0.109 mL),
Vent(exoꢀ) (2 UmLꢀ1 0.109 mL), Vent(exoꢀ) (2 UmLꢀ1
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ˇ
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9410, Amersham Biosciences).
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,
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DTT (5 mm), MgCl2 (5 mm), all four natural dNTPs (100 mm) and the
modified dNTP (11c-TP, 11g-TP or 11h-TP) at the given concentra-
tion (5–900 mm). The reactions were carried out at 378C for 60 min
and were stopped by adding 20 mL PAGE stop solution and heating
at 958C for 5 min. Aliquots (4 mL) were subjected to electrophoresis
in 12.5% denaturing polyacrylamide gel.
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Acknowledgements
This work was supported by the Academy of Sciences of the
Czech Republic (RVO: 61388963), the Czech Science Foundation
(P207/11/0344), and by Gilead Sciences Inc.
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Keywords: 7-deazapurines · antiviral agents · cytostatics ·
fluorinated derivatives · nucleosides
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Received: January 31, 2013
Published online on April 4, 2013
ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 832 – 846 846