Chemo-enzymatic synthesis and biological evaluation of 5,6-disubstituted benzimidazole ribo- and 2′-deoxyribonucleosides

Article abstract of DOI:10.1055/s-0032-1317782

A number of new 5,6-disubstituted benzimidazoles have been prepared and their substrate properties for recombinant E. coli purine nucleoside phosphorylase (PNP; the product of the deoD gene) in the transglycosylation reaction were investigated. The heterocyclic bases showed good substrate activity for PNP and the ribo- and 2-deoxyribonucleosides were synthesized. The predominant (OMe and OEt) or exclusive (Oi-Pr, morpholino, and N-methylpiperazino) formation of the 5-substituted 6-fluoro-1-(β-d- ribofuranosyl)benzimidazoles was observed. The formation of the regioisomeric 6- methoxy-, 6-ethoxy-, or 6-isopropoxy-substituted 1-(2-deoxy-β-d- ribofuranosyl)-5-fluorobenzimidazoles was observed in the trans-2- deoxyribosylation reaction of the corresponding bases. The predominant or exclusive formation of the regioisomeric N¹-nucleosides with bulky 5-substituents of 6-fluorobenzimidazole points to a large hydrophobic pocket in the E. coli PNP active site that can accommodate these groups. The biological activity of the synthesized nucleosides was studied and revealed no inhibitory activity against a broad variety of DNA and RNA viruses. The compounds also lacked significant cytotoxicity. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0032-1317782

272▌
PAPER
paper
Chemo-Enzymatic Synthesis and Biological Evaluation of 5,6-Disubstituted
Benzimidazole Ribo- and 2′-Deoxyribonucleosides
Chemo-Enzymatic Syntheses and Biological Evaluation
Irina D. Konstantinova,a Olga M. Selezneva,a Ilja V. Fateev,a Tamara A. Balashova,a Svetlana K. Kotovskaya,b
Zoya M. Baskakova,b Valery N. Charushin,b Alexander V. Baranovsky,c Anatoly I. Miroshnikov,a Jan Balzarini,*d
Igor A. Mikhailopulo*c
a
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP,
Moscow B-437, Russian Federation
b
I. Ya. Postovsky Institute of Organic Synthesis, the Ural Branch of the Russian Academy of Sciences, Sophia Kovalevskaya/Academicheskaya
St. 22/20, 620041 Yekaterinburg, Russian Federation
c
Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus
Fax +375(17)2678761; E-mail: imikhailopulo@gmail.com
d
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
Received: 11.10.2012; Accepted after revision: 16.11.2012
pholino, or N-methylpiperazino group, and their use as ac-
Abstract: A number of new 5,6-disubstituted benzimidazoles have
ceptors of D-ribofuranose and 2-deoxy-D-ribofuranose
been prepared and their substrate properties for recombinant E. coli
residues in transglycosylation reactions. Uridine (or 2′-de-
oxyuridine) and thymidine are employed as the pentofura-
purine nucleoside phosphorylase (PNP; the product of the deoD
gene) in the transglycosylation reaction were investigated. The
heterocyclic bases showed good substrate activity for PNP and the nose donors and recombinant E. coli uridine (UP; EC
ribo- and 2′-deoxyribonucleosides were synthesized. The predomi-
nant (OMe and OEt) or exclusive (Oi-Pr, morpholino, and N-meth-
2.4.2.3), thymidine (TP; 2.4.2.4), and purine (PNP; product
of deoD gene; EC 2.4.2.1) nucleoside phosphorylases11
ylpiperazino) formation of the 5-substituted 6-fluoro-1-(β-D-
were used as the biocatalysts (see Scheme 2).
ribofuranosyl)benzimidazoles was observed. The formation of the
Synthesis of 5,6-difluoro-1H-benzimidazole (1) was real-
ized from 4,5-difluoro-2-nitroaniline (2)12 by the catalytic
reduction of the nitro group (H2, Raney Ni),13 followed by
the well-documented imidazole ring-closure reaction by
treatment with triethyl orthoformate. It was found that
fluorine atoms of difluoride 1 are very resistant to nucleo-
philic substitution and attempts to replace one of them by
regioisomeric 6- methoxy-, 6-ethoxy-, or 6-isopropoxy-substituted
1-(2-deoxy-β-D-ribofuranosyl)-5-fluorobenzimidazoles was ob-
served in the trans-2-deoxyribosylation reaction of the correspond-
ing bases. The predominant or exclusive formation of the
regioisomeric N1-nucleosides with bulky 5-substituents of 6-fluoro-
benzimidazole points to a large hydrophobic pocket in the E. coli
PNP active site that can accommodate these groups. The biological
activity of the synthesized nucleosides was studied and revealed no
inhibitory activity against a broad variety of DNA and RNA viruses. an alkoxy group failed. Therefore, the sequence of reac-
The compounds also lacked significant cytotoxicity.
tions depicted in Scheme 1 was used to synthesize mono-
fluorides 5–9 starting from the difluoride 2. The physico-
chemical data for compounds 1, 5–9 are given in the ex-
perimental section and Table 2.
Key words: 5,6-disubstituted benzimidazoles, purine nucleoside
phosphorylase, substrate properties, transglycosylation reaction,
nucleosides
The transglycosylation reaction was employed for the
synthesis of β-D-ribo- and 2-deoxy-β-D-ribonucleosides
10–21 (Scheme 2). The reaction conditions were opti-
mized depending on the ribofuranose donor, the donor/
base ratio, quantity of the recombinant E. coli enzymes,
and temperature of the reaction. The use of readily avail-
able natural purine ribonucleosides as donors of the ribo-
furanose residue in the transglycosylation reaction of
benzimidazoles allows PNP to be employed as the sole
biocatalyst. In contrast, the use of uridine (2′-deoxyuri-
Halogenated benzimidazole-based nucleosides have at-
tracted much attention since the pioneering studies by
Tamm and co-workers initiated in the early 1950s.1 How-
ever, the most important findings from the viewpoint of
the biochemical mechanism of antiviral activity of this
class of modified nucleosides2 as well as their possible
practical application3 were published over the last two de-
cades.4,5
It was earlier shown that benzimidazole and its derivatives dine) or thymidine as donors of the pentofuranose resi-
with substituents in the benzene ring are good substrates dues requires two nucleoside phosphorylases for
of E. coli PNP in transglycosylation reactions.6–10 In the transglycosylation reactions, viz., recombinant E. coli UP
present study, we report on the synthesis of 5,6-difluoro- or TP for the intermediary formation of α-D-ribofuranose
benzimidazole and its derivatives, where one fluorine 1-phosphate (α-D-Rib-1P) or 2-deoxy-α-D-ribofuranose
atom is replaced by a methoxy, ethoxy, isopropoxy, mor- 1-phosphate (α-D-dRib-1P) that is accepted by E. coli
PNP for the synthesis of the benzimidazole nucleosides.
The efficiency of natural purine vs. pyrimidine ribonu-
cleosides as donors was tested in the transglycosylation of
the difluoride 1 and it was found that the use of uridine (2′-
deoxyuridine) or thymidine and the respective UP or TP
SYNTHESIS 2013, 45, 0272–0280
Advanced online publication: 07.12.2012
DOI: 10.1055/s-0032-1317782; Art ID: SS-2012-T0784-OP
© Georg Thieme Verlag Stuttgart · New York
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X

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Chemo-Enzymatic Syntheses and Biological Evaluation
273
F
F
N
N
H
1
O2N
H2N
F
F
O2N
H2N
F
H2N
H2N
F
F
N
N2H4
R-H
CH(OEt)3
N
H
Ni-Raney
R
R
R
5. R = OMe
6, R = OEt
7, R = Oi-Pr
2
3
4
R = OMe (a); OEt (b); Oi-Pr (c); morpholino
(d); N-methylpiperazino (e)
8, R = morpholino
9, R = N-methylpiperazino
Scheme 1 Structure of 5,6-difluoro-1H-benzimidazole (1) and synthesis of 6(5)-substituted 5(6)-fluorobenzimidazoles 5–9
Urd(dUrd)/UP
or
base (1,5–9)
PNP
O
P
F
R
F
HO
N
N
Pi
O
+
OH
O
– Ura or Thy
– Pi
N
N
R
O
Thd/TP
Pfr
Pfr
HO
X (OH; H)
a
b
Scheme 2 Enzymatic synthesis of 5,6-disubstituted benzimidazole ribo- and 2′-deoxyribo-nucleosides 10–21
nucleoside phosphorylases in combination with PNP is ranose 1-phosphate (Table 1, for experimental data on the
preferable in terms of yield of the desired nucleosides enzymatic synthesis see Table 3, and physico-chemical
(see, e.g., Figure 1).
properties of nucleosides synthesized, see Tables 4 and 5).
Reactions were conducted at 52 °C in potassium phos-
phate buffer (5–20 mM; pH 7.0) monitoring the formation
of the products by HPLC. The conversion of base into nu-
cleoside(s) was ≥98.4%. Compounds 10 and 11 were
crystallized directly from the reaction mixtures, and the
other nucleosides were isolated by C18 RP silica gel col-
umn chromatography. It is noteworthy that the synthesis
of 2′-deoxyribosides was completed in 1–3 hours, where-
as the transribosylation reaction proceeded more slowly
and required 22–28 hours to achieve high yields of prod-
ucts (Table 3). A similar trend was earlier observed in the
case of the ribo- and 2′-deoxyribonucleoside synthesized
using whole E. coli cells as the biocatalyst.7
Figure 1 Progress of the synthesis of the riboside 10 depending on
the type of D-ribofuranose donor (Ino = inosine; Urd = uridine;
Guo = guanosine; Ado = adenosine)
The regioisomeric structure of all isolated nucleosides
was proven by scrupulous analysis of the 1H and 13C NMR
spectral data (including [1H,1H] and [1H,13C] 2D: COSY,
HSQC, HMBC, and NOE spectra) (see Table 5).
Furthermore, the reaction conditions were optimized with
regard to the donor/base ratio, quantity of the recombinant
E. coli enzymes, and temperature of the reaction mixture.
It was found that the 3:1 to 10:1 molar donor/base ratio
and the use of 40 units of uridine phosphorylase (UP) per
1 mmol of uridine (60–160 UP units for 2′-deoxyuridine)
and 155–400 units of PNP afforded the ribosides 10–21 in
good yields calculated for the isolated products. In a sim-
ilar way, thymidine was used for the synthesis of 2′-de-
oxyribosides 19 and 21 as a donor of the 2-deoxy-D-
ribofuranose residue and thymidine phosphorylase (TP)
for the generation of the intermediate 2-deoxy-α-D-ribofu-
Thus, for example in the [1H,1H] NOESY spectrum of the
riboside 18a, methylene N(CH2)2 protons (δ = 2.98)
showed strong interaction with the proton at C4 (δ = 7.31,
d), whereas the proton at C7 (δ = 7.70, d) interacted with
the H2′, H1′, and H5′ protons (δ = 4.30, 5.79, and 3.65, re-
spectively) (Figure 2). These observations allow the for-
mer doublet to be unequivocally assigned to H4 [4JH4,F6
=
7.9 Hz; C4 δ = 108.92 (s, 3JC4,F6 < 1.0 Hz)] and the latter to
H7 [3JH7,F6 = 12.4 Hz; C7 δ = (99.19, d, 2JC7,F6 = 27.9 Hz)],
as well as the position of the fluorine atom at C6. More-
over, the values of the ortho F,H7 (3JH7,F6 = 12.4 Hz) and
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 272–280

274
I. D. Konstantinova et al.
PAPER
guanines as substrates in the transglycosylation reac-
tions.15 In contrast, E. coli PNP cannot tolerate guanine
derivatives with such bulky and voluminous functions as
N2-palmitoyl and O6-diphenylcarbamoyl. More recently,
we reported the use of N2-acetyl-O6-[2-(4-nitrophe-
nyl)ethyl]-guanine as a good substrate for E. coli PNP in
transglycosylation reactions.16 These data taken together
enable one to visualize the spatial dimensions of a hydro-
phobic pocket in vicinity of the C6–N1–C2 segment of the
pyrimidine ring of purines and the C5 atom of benzimid-
azole derivatives in the E. coli PNP active site that can ac-
commodate these groups (Figure 3).
O
H
4
N
N
N
H
F
7
HO
H
5'
O
H
H
1'
H
OH 2O' H
H
3'
Figure 2 The observed NOE effects of protons H4 and H7 of the ri-
boside 18a
meta F,H4 (4JH4,F6 = 7.94 Hz) couplings are in favor of the
suggested structure 18a (cf. ref.14). The 13C resonances of
C4 and C7 carbon atoms arise from the [1H,13C] 2D spec-
tra. A similar approach was used for the assignment of the
structures of other nucleosides.
Table 1 Yields of the Isomeric Nucleosides
Figure 3 The geometry optimized (HyperChem, 8.1; the ab initio
method, in vacuo, basis set; medium 6-31G* level) structures of N2-
acetyl-O6-[2-(4-nitrophenyl)ethyl]guanine (left), 5-morpholino-6-
fluoro-1H-benzimidazole (right), and an overlay of the imidazole
fragments of both structures (middle)
Compd Pfra
R
Yieldb (%) Isomer ratio
a
b
10
Rib
dRib
77
68
–
–
–
–
F
11
12
13
14
15
16
17
Remarkably, the pocket in the E. coli PNP catalytic site
exceeds the dimension of the purine base and allows a
minimal contribution of the C6 amino/carbonyl groups
and N1 nitrogen atom to the substrate binding to be sug-
gested.
Rib
dRib
54
51
95
41
5
59
OMe
OEt
Oi-Pr
Rib
dRib
98
85
89
70
11
30
None of the compounds were cytostatic in cell culture,
and they also did not show significant inhibitory activity
against the replication of a broad variety of DNA and
RNA viruses.
Rib
dRib
75
65
100
96
–
4
O
N
18
19
Rib
dRib
75
80
100
100
–
–
N
In conclusion, the efficient enzymatic method for the
preparation of the 5-substituted 6-fluoro-1-(β-D-ribofura-
nosyl)- and 1-(2-deoxy-β-D-ribofuranosyl)-6-fluorobenz-
imidazoles is described. 5,6-Difluoro-1H-benzimidazole
(1)12,13 was found to be inert to nucleophilic substitution
of a fluorine atom and another sequence of chemical
transformations was, therefore, used for the synthesis of
the desired heterocyclic bases 5–9, viz., replacement of
one of the fluorine atoms of the starting 4,5-difluoro-2-ni-
troaniline (2)12 with a nucleophilic reagent, conventional
reduction of the nitro group into an amine, and imidazole
ring closure by treatment with triethyl orthoformate fur-
nished the bases 5–9 in good yields.
Me
20
21
Rib
dRib
79
68
100
100
–
–
N
a Pfr = β-D-pentofuranosyl (Rib = β-D-ribofuranosyl; dRib = 2′-de-
oxy-β-D-ribofuranosyl).
b Isolated yield.
The regioselectivity of the transglycosylation reaction in
the case of bases 7, 8, and 9 is of interest from the view-
point of the structure of the catalytic site of E. coli PNP. It
is obvious that the bulkiness of the respective isopropoxy,
morpholino, and N-methylpiperazino substituents is the
main reason for the strict regioselectivity observed.
The benzimidazole derivatives 1, 5–9 showed good sub-
strate activity for E. coli PNP in transglycosylation reac-
tions. The use of uridine (2′-deoxyuridine)/UP or
thymidine/TP is favorable vs. natural purine nucleosides
and PNP for the production of intermediate 1-phosphates
of α-D-ribo- or α-D-2-deoxyribofuranoses. The transgly-
cosylation of bases 1, 5–9 under optimal reaction condi-
tions resulted in a more than 98.4% conversion of the base
into nucleosides. It is notable that rather low rate of the
The predominant or exclusive formation of the regioiso-
meric N1-nucleosides 16–21 with bulky 5-substituents on
6-fluorobenzimidazole points to a large hydrophobic
pocket allowing the respective bases to bind in the E. coli
PNP catalytic site and to realize the coupling of base and
pentofuranose. We have previously shown that E. coli
PNP accepts kinetin, N6-benzoyl- and N6-benzyl-adenines
as well as N2-acetyl-O6-methyl- and N2-acetyl-O6-benzyl-
Synthesis 2013, 45, 272–280
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Chemo-Enzymatic Syntheses and Biological Evaluation
275
4-Fluoro-5-(N-methylpiperazino)-2-nitroaniline (3e)
transribosylation (22–28 h) vs. that of trans-2-deoxyribo-
sylation of benzimidazoles and arabinosylation of 2-fluo-
roadenine (1–3 h),17 are in accord with results of the ab
initio geometry optimization (HyperChem, 8.1 release; in
vacuo, 6-31G* level) of the spatial structures of the corre-
sponding 1-phosphates.18 In chemical terms, the conden-
Yellow crystals; yield: 3.51 g (46%); mp 136–137 °C; Rf = 0.54.
5-Substituted 4-Fluorobenzene-1,2-diamines 4a–e; General
Procedure
A mixture of compound 3 (30 mmol), hydrazine hydrate (98%, 4
mL) and Raney Ni (89%, 5% of the weight compound 3) in EtOH
(50 mL) was refluxed for 4–5 h. The hot reaction mixture was fil-
tered, the residue on the filter was washed with EtOH (30 mL) and
combined filtrate and washing was stored at +4 °C. The dark-brown
sation
of
α-D-pentofuranose-1-phosphates
with
heterocyclic bases occurs as a result of nucleophilic attack
of the base nitrogen atom on the electrophilic anomeric precipitate was filtered off, washed with EtOH and dried.
carbon atom of the 1-phosphate. Indeed, the calculated
values of the partial positive charges of the C1 atoms of α-
4-Fluoro-5-methoxybenzene-1,2-diamine (4a)
Colorless crystalline powder; yield: 3.04 g (65%); mp 110–112 °C;
D-ribo- (0.425 e), α-D-2-deoxyribo- (0.454 e), and α-D-
arabinofuranosyl 1-phosphates (0.464 e) (suggest increas-
ing electrophilic properties of the anomeric carbon atom
among these compounds.
Rf = 0.65.
5-Ethoxy-4-fluorobenzene-1,2-diamine (4b)
Colorless crystalline powder; yield: 3.21 g (63%); mp 80–82 °C;
Rf = 0.56.
One of the most important results of this work is that we
were able to provide the visual size of the hydrophobic
pocket of E. coli PNP. This result allows us to understand
the mechanism of substrate binding in the active site of the
enzyme, the subsequent activation of the substrate, and
the formation of the glycosidic bond.18
4-Fluoro-5-isopropoxybenzene-1,2-diamine (4c)
Brown powder; yield: 1.58 g (35%); mp 130–132 °C; Rf = 0.52
4-Fluoro-5-morpholinobenzene-1,2-diamine (4d)
Cream powder; yield: 3.42 g (54%); mp 126–127 °C; Rf = 0.49
4-Fluoro-5-(N-methylpiperazino)benzene-1,2-diamine (4e)
Cream powder; yield: 3.03 g (45%); mp 78–80 °C; Rf = 0.46.
Hydrazine hydrate and Raney Ni were purchased from Sigma-
Aldrich. Silica gel C18 was supplied by Merck. NMR spectra:
Bruker Avance-500-DRX and Bruker Avance-700-DRX (Bruker,
Germany). Mass spectra: Agilent 6224, ESI-TOF, LC/MS (USA)
and Esquire 3000 Plus (Bruker Daltonics, Germany) in positive ion
mode (ESI+). HPLC was performed on Waters system (Waters
1525, Waters 2487, Breeze 2; USA); column: Nova Pack C18, 4
μm, 4.6 × 150 mm; eluent А: 0.1% TFA–H2O, eluent В: 70%
MeCN in 0.1% TFA–H2O; flow: 1 mL/min; UV detection: 254 and
280 nm. The UV spectra were recorded on the UV spectrophotom-
eter Shimadzu UV-160 (Japan). Progress of the synthesis of com-
pounds 3 and 4 and their purity was monitored and checked,
respectively, by TLC [Sorbophil (Merck), CHCl3–EtOH, 5:1]; sat-
isfactory C, H and N (±0.40) elemental analyses were obtained for
compounds 3.
5-Substituted 6-Fluorobenzimidazoles 5–9; General Procedure
Compound 4 (30 mmol) was refluxed in CH(OEt)3 (60 mL) for 6–
7 h, the mixture was evaporated to ca. 10 mL and residue poured out
into cold (3–5 °C) H2O. The precipitate formed was filtered off,
dried and recrystallized (EtOH–H2O). Physico-chemical and NMR
data are collected in Table 2.
5,6-Difluoro-1H-benzimidazole (1)
Yellow powder; yield: 4.02 g (87%); mp 178–179 °C.
5-Fluoro-6-methoxy-1H-benzimidazole (5)
Colorless crystals; yield: 1.99 g (40%); mp 86–87 °C.
5-Ethoxy-6-fluoro-1H-benzimidazole (6)
Colorless crystals; yield: 3.24 g (60%); mp 98–100 °C.
5-Fluoro-6-isopropoxy-1H-benzimidazole (7)
Yellow oil; yield: 1.68 g (35%).
The preparation of recombinant E. coli UP, TP and PNP (the prod-
uct of the deoD gene) was described previously.11
5-Fluoro-6-morpholino-1H-benzimidazole (8)
5-Alkoxy-4-fluoro-2-nitroanilines 3a–c; General Procedure
To a soln of 4,5-difluoro-2-nitroaniline12 (2, 5.22 g, 30 mmol) in the
alcohol (50 mL) was added a soln of NaOH (30 mmol) in the same
alcohol and the mixture was stirred at r.t. for 1 h. The precipitate
formed was filtered off, dried, and recrystallized (EtOH).
Cream crystalline powder; yield: 5.31 g (80%); mp 208–210 °C.
5-Fluoro-6-(N-methylpiperazino)-1H-benzimidazole (9)
Cream crystals; yield: 2.81 g (40%); mp 120–122 °C.
Enzymatic Reactions
The enzymatic reactions were performed in potassium phosphate
buffers (5–20 mM; pH 7.0) at 52 °C monitoring the reaction prog-
ress by HPLC; the reactions were stopped by addition of EtOH
when >98.5% of base was transformed into nucleoside. The mixture
was evaporated to dryness and compounds 10 and 11 were recrys-
tallized (H2O). The remaining products were purified by column
chromatography (silica gel C18, 20 × 120 mm, H2O–MeCN). For
details see Table 3 and physico-chemical and spectral data in Tables
4 and 5.
4-Fluoro-5-methoxy-2-nitroaniline (3a)
Yellow crystals; yield: 4.81 g (80%); mp 169–170 °C; Rf = 0.82.
5-Ethoxy-4-fluoro-2-nitroaniline (3b)
Yellow crystals; yield: 5.10 g (85%); mp 134–135 °C; Rf = 0.86.
4-Fluoro-5-isopropoxy-2-nitroaniline (3c)
Yellow crystals; yield: 1.93 g (30%); mp 150–152 °C; Rf = 0.78.
4-Fluoro-5-(morpholino)- (3d) and 4-Fluoro-5-(N-methylpiper-
azino)-2-nitroaniline (3e)
A mixture of 4,5-difluoro-2-nitroaniline (2; 30 mmol) and morpho-
line (60 mmol) or N-methylpiperazine (60 mmol) in EtOH (25 mL)
was refluxed for 30–60 min and cooled to r.t. The precipitate
formed was filtered off, dried and recrystallized (EtOH).
Antiviral Assays
The antiviral assays other than the anti-HIV assays, were based on
inhibition of virus-induced cytopathicity in HEL [herpes simplex
virus type 1 (HSV-1), HSV-2 (G), vaccinia virus, vesicular stoma-
titis virus, cytomegalovirus (HCMV) and varicella-zoster virus
(VZV)], Vero (parainfluenza-3, reovirus-1, Sindbis virus and Cox-
sackie B4), HeLa (vesicular stomatitis virus, Coxsackie virus B4,
and respiratory syncytial virus), Crandel–Rees feline kidney
4-Fluoro-5-(morpholino)-2-nitroaniline (3d)
Yellow crystals; yield: 4.56 g (63%); mp 188–190 °C; Rf = 0.65.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 272–280

276
I. D. Konstantinova et al.
PAPER
(CRFK) [feline coronavirus (FIPV) and feline herpes virus] or
MDCK [influenza A (H1N1; H3N2) and influenza B] cell cultures.
Confluent cell cultures (or nearly confluent for MDCK cells) in mi-
crotiter 96-well plates were inoculated with 100 CCID50 of virus (1
CCID50 being the virus dose to infect 50% of the cell cultures) in the
presence of varying concentrations of the test compounds. Viral cy-
topathicity was recorded as soon as it reached completion in the vi-
rus-infected cell cultures that were not treated with the test
compounds.
Anti-HIV Assays
Human lymphocyte CEM cells (~3 × 105 cells/mL) cells were in-
fected with 100 CCID50 of HIV(IIIB) or HIV-2(ROD)/mL and seed-
ed in 200 μL wells of a microtiter plate containing appropriate
dilutions of the test compounds. After 4 d of incubation at 37 °C, vi-
rus-induced cytopathicity (giant cell formation) was recorded mi-
croscopically. The EC50 was defined as the compound
concentration required to inhibit virus-induced cytopathogenicity
by 50%. The CC50 was defined as the compound concentration re-
quired to inhibit CEM cell proliferation by 50%.
Table 2 Yields and 1H NMR Data for Compounds 1, 5–9
Compd Yielda (%) 1H NMR (DMSO-d6) δ, J (Hz)
H2 (s) H4
H7
NH (br s) Other
1
5
6
7
87
40
60
35
8.15
7.44 (m, 2 H)
12.11
12.25
12.00
12.15
–
7.95
7.94
7.95
7.16 (m)
7.13 (m)
7.16 (m)
7.28 (d, J = 11.0)
7.27 (d, J = 10.8)
7.27 (d, J = 11.0)
3.87 (s, 3 H, OCH3)
1.43 (t, 3 H, OCH2CH3), 4.08 (dd, 2 H, OCH2CH3)
1.33 [d, 6 H, OCH(CH3)2], 4.50 [m, 1 H,
OCH(CH3)2]
8
9
80
40
7.94
7.94
7.11 (d, J = 7.8) 7.24 (d, J = 12.5)
7.20 (m) 7.24 (d, J = 12.0)
12.00
12.00
2.46–2.50 (t, 4 H, CH2OCH2), 2.98–3.01 (t, 4 H,
CH2NCH2)
2.26 (s, 3 H, NCH3), 2.50 (m, 4 H, CH2NCH2), 2.99
(m, 4 H, CH2NCH2)
a Yields of isolated pure compounds; satisfactory C, H, and N (±0.40) elemental analyses were obtained.
Table 3 Experimental Data for the Enzymatic Synthesis of Benzimidazole β-D-Ribo- and β-D-2′-Deoxyribonucleosidesa
Compd Acceptorb Donorb Substrates
PNPc units UPc units TPc units Time Residual MeCNd
(A)
(B)
(C)
(h)
base (%) (%)
Acceptor
Donor
Ratio
(mg) [mmol] (mg) [mmol] A/D
10
11
12
13
14
15
16
17
18
19
20
21
1
1
5
5
6
6
7
7
8
8
9
9
Urd
100 [0.65]
200 [1.3]
180 [1.08]
56 [0.34]
200 [1.1]
180 [1.0]
40 [0.2]
474 [1.94]
887 [3.9]
1:3
1:3
101 (155) 78 (40)
207 (160) 233 (60)
169 (155) 432 (40)
–
22
1
1.49
1.09
0.26
0.42
1.57
0.86
0.78
1.10
0.96
1.07
1.32
1.08
0
dUrd
Urd
–
0
2.647 [10.8] 1:10
–
26
3
0
dUrd
Urd
771 [3.38]
1.355 [5.5]
1:10
1:5
53 (155)
304 (90)
–
20
10
10
21
25
0
177 (160) 230 (40)
160 (160) 460 (80)
–
24
3
dUrd
Urd
2.282 [10.0] 1:10
–
503 [2.0]
470 [2.0]
1.545 [6.3]
2.276 [9.4]
415 [1.7]
288 [1.2]
1:10
1:10
1:7
80 (400)
80 (400)
80 (40)
–
24
2
dUrd
Urd
40 [0.2]
315 (160)
–
200 [0.9]
297 [1.3]
40 [0.17]
40 [0.17]
144 (160) 250 (40)
–
24
1
Thd
Urd
1:7
260 (200)
51 (300)
68 (400)
–
891 (95)
–
10
20
25
1:10
1:7
68 (40)
–
28
Thd
128 (107) 1
a For conditions, see the experimental procedures.
b MW used: 1 (154.12); 5 (166.16); 6 (180.19); 7 (194.21); 8 (221.24); 9 (234.25); Urd (244.2); dUrd (228.2); Thd (242.23).
c A: ratio of PNP (units)11 per acceptor base (1 mmol), e.g., 101/0.65 = 155; B: ratio of UP (units)11 per nucleoside [uridine (Urd) or 2′-
deoxyuridine (dUrd)] (1 mmol), e.g., 78/1.94 = 40; C: ratio of TP (units)11 per thymidine (Thd; 1 mmol), e.g., 891/9.4 = 95.
d Nucleosides were eluted from the C18-silica gel column with a MeCN–H2O mixture, the percentage of MeCN is given.
Synthesis 2013, 45, 272–280
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277
Table 4 Physicochemical Properties of Nucleosides 10–21
Compound
Yield (%) Purity (%)
[mg]
[HPLC]a
Mp (°C)
UVc
Calcd
MW
MSd
(solvent)b
λmax (nm) [ε]
R
F
Pfr
10
11
Rib 77 [143.1] 99.89
[tR = 8.9 (II)]
186–190
(H2O)e
278.4 [5,200],
243.4 [6,700]
286.233 286.838 [M + H]+, 154.711
[M – Rib + 2 H]+, 115.707
[M – Rib – 2 F + H]+
F
dRib 68 [238.7] 99.67
[tR = 9.4 (II)]
175–178
(H2O)e
278.8 [5,700],
243.2 [7,100]
270.233 271.071 [M + H]+,
154.9281 [M – dRib + 2
H]+, 116.912 [M – B]+
12a (ratio
12a/12b 95:5)
OMe
OMe
Rib 54 [173.8] 97.62
[tR = 14.05 (I)]
–
–
298.268 299.097 [M + H]+
13a,b
dRib 51 [48.9]
13a: 40.90
282.269 283.022 [M + H]+
(ratio 13a/13b
41:59)
[tR = 15.37 (I)]
13b: 59.10
[tR = 14.07 (I)]
14a
(ratio 14a/14b
89:11)
OEt
OEt
Rib 98 [336.4] 100
[tR = 9.24 (III)]
–
–
312.295 313.115 [M + H]+, 180.976
[M – Rib + 2 H]+
15a
(ratio 15a/15b
70:30)
dRib 85 [251.6] 100
[tR = 8.00 (II)
296.296 297.064 [M + H]+, 180.963
[M – dRib + 2 H]+, 115.889
[M – dRib – F – C2H5O +
H]+
16a
Oi-Pr
Oi-Pr
Rib 75 [48.9]
dRib 65 [40.3]
97.88
[tR = 9.57 (III)]
66–69
289.6 [4,800],
247.4 [5,200],
204.8 [32,400]g
326.322 327.080 [M + H]+, 194.880
[M – Rib + 2 H]+
f
17a
100
–
–
310.322 311.044 [M + H]+
(ratio 17a/17b
[tR = 14.79 (V)]
96:4)
18a
19a
morpholino Rib 74 [235.2] 100
[tR = 12.92 (IV)]
92–96
290.2 [5,550],
214.6 [20,700]
353.347 354.166 [M + H]+, 221.026
[M – Rib + H]+, 222.024
[M – Rib + 2 H+], 334.144
[M – F]+
morpholino dRib 80 [350.6] 98.86
[tR = 14.08 (IV)]
154–158
290.8 [6,050],
213.8 [21,400]
{295.2 [6,450],
219.0 [20,900]h}
337.348 338.163 [M + H]+, 318.136
[M – F]+, 221.025 [M –
dRib + H]+, 222.016 [M –
dRib + 2 H]+
20a
21a
N-methyl- Rib 79 [49.2]
piperazino
100
[tR = 5.76 (III)]
104–108
84–88
288.2 [6,600],
214.0 [28,000]
336.389 367.175 [M + H]+, 234.993
[M – Rib + 2 H]+
N-methyl- dRib 68 [40.47] 94.58
piperazino [tR = 6.35 (III)]
288.2 [6,500],
213.6 [26,600]
350.390 351.114 [M + H]+, 234.958
[M – dRib + 2 H]+, 213.779
[M – dRib – F]+, 214.780
[M – dRib – F + H]+
a HPLC (Waters 1525, Waters 2487, Breeze 2; column: Nova Pack C18, 4 μm, 4.6 × 150 mm; eluent А: 0.1 % ТFA–H2O, eluent В: 70%
MeCN in 0.1 % ТFA–H2O; flow: 1 mL/min; UV detection: 254 and 280 nm). Elution: (I) 7% В; (II) 7% В over 5 min, 7% to 100% В over
15 min; (III) 0 to 35% В over 10 min, then 35% В over 10 min; (IV) 7% В over 5 min, 7% to 15% В over 1 min, then 15% В over 14 min;
(V) 7% В over 5 min, 7% to 20% В over 2 min, then 20% В over 10 min.
b White amorphous powder unless otherwise stated.
c UV spectra recorded on a UV-spectrophotometer Shimadzu UV-160 (Japan) in H2O, unless otherwise indicated.
d The mass spectra [electrospray ionization (ESI)] were recorded on Esquire 3000 Plus (Bruker Daltonics, Germany).
e Colorless crystals.
f Colorless oil.
g In EtOH.
h Aq 80% EtOH.
© Georg Thieme Verlag Stuttgart · New York
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278
I. D. Konstantinova et al.
PAPER
Table 5 Selected 1H and 13C NMR Spectroscopy Data of Benzimidazole Nucleosides (DMSO-d6) δ, J (Hz)a
Compd H1′
[C1′]
H2′a
[C2′]
H2′b
[C2′]
H3′
[C3′]
H4′
[C4′]
H5′a/H5′b Other [13C]
[C5′]
10
5.86 (d, J = 4.33 (m, J
–
4.12 (m)
[70.47]
4.00 (m)
[86.16]
3.66 (m) 8.52 (1 H, H2) [144.53], 8.01 (dd, 3JH4,F5 = 10.8,
6.6)
= 6.6, 5.6)
[73.84]
[61.55]
4JH4,F6 = 7.5, 1 H, H4) [100.39], 7.75 (dd, 3JH7,F6
=
[89.39]
11.0, 4JH7,F5 = 7.5, 1 H, H7) [106.91], 5.41 (br s, 2′-
OH), 5.19 (br s, 3′-OH), 5.26 (br s, 5′-OH)
11
6.34 (dd,
2.54 (m, 2.26 (octet, 4.40 (dt, J = 3.88 (q, J = 3.58 (m) 8.54 (1 H, H2) [144.04], 7.98 (dd, 3JH4,F5 = 10.8,
J = 7.4, 6.2) gemJ=13.6, gemJ = 13.2, 6.3, 3.2)
3.7)
[–b]
4JH4,F6 = 7.4, 1 H, H4) [100.07 (d, 3JC4,F5 = 23.5,
C4)], 7.73 (dd, 4JH7,F5 = 7.5, 3JH7,F6 = 11.0, 1 H, H7)
[106.83 (d, 3JC7,F6 = 19.4)], 5.36 (d, J = 4.0, 1 H, 3′-
OH), 5.07 (t, J = 5.2, 1 H, 5′-OH)
[–b]
7.5, 6.30 5.9, 3.0)
[–b]
[–b]
[–b]
[–b]
–
12a
12b
13a
13b
14a
5.79 (d, J = 4.31 (dd)
6.4)
[88.55]
4.11 (br s) 3.97 (br m) 3.63, 3.67 8.36 (s, 1 H, H2) [142.46], 7.76 (d, 3JH7,F6 = 11.4, 1
[69.70] [85.17]
(dd, dd) H, H7) [98.91 ], 7.41 (d, 4JH4,F6 = 7.8, 1 H, H4)
[73.00]
[60.88]
[103.11 ], 5.39 (br s, 2′-OH), 5.17 (br s, 3′-OH), 5.15
(br s, 5′-OH), 3.86 (s, 3 H, ОCH3) [55.99 (s)]
5.85 (d, J = 4.38 (dd)
6.5)
[88.49]
–
4.13 (br s) 3.99 (br m) 3.63, 3.67 8.33 (s, 1 H, H2) [142.22], 7.64 (d, 4JH7,F5 = 7.7, 1 H,
[72.92]
[69.78]
[85.25]
(dd, dd) H7) [96.11 ], 7.50 (d, 3JH4,F5 = 11.6, 1 H ,H4)
[60.88]
[105.28 ], 5.44 (br s, 2′-OH), 5.19 (br s, 3′-OH), 5.16
(br s, 5′-OH), 3.86 (s, 3 H, ОCH3) [55.99]
6.29 (dd,
J = 7.3, 6.4) [39.28]
[84.43]
2.55 (m) 2.25 (m)
4.40 (m)
[70.28]
3.86 (m)
[87.36]
3.62–3.58 8.38 (s, 1 H, H2) [141.82], 7.70 (d, 3JH7,F6 = 11.5, 1
(m)
[61.12]
H, H7) [98.67 ], 7.39 (d, 4JH4,F6 = 7.9, 1 H, H4)
[103.11 ], 5.30 (d, J = 4.1, 3′-OH), 4.98 (t, J = 5.1,
5′-OH), 3.87 (3 H, ОCH3) [56.07]
6.36 (dd,
J = 7.3, 6.5) [39.31]
[84.40]
2.56 (m) 2.26 (octet, 4.43 (m)
3.88 (m)
[87.15]
3.61–3.59 8.36 (s, 1 H, H2) [141.56], 7.57 (d, 3JH7,F5 = 7.9, 1 H,
(m)
[61.18]
gemJ = 13.1, [70.21]
H7) [95.83], 7.49 (d, 4JH4,F5 = 11.7, 1 H, H4) [105.26
], 5.31 (d, J = 3.9 Hz, 3′-OH), 5.01 (t, J = 5.2, 5′-
OH), 3.89 (3 H, ОCH3) [56.10]
5.8, 2.7)
5.79 (d, J = 4.31 (dd)
6.4)
[88.75]
–
4.11 (br m, 3.97 (m)
3.61–3.69 8.35 (s, 1 H, H2) [142.5], 7.74 (d, J = 11.4, 1 H, H7)
(m, gemJ = [99.15 (d, 2JC7,F6 = 25.17)], 7.39 (d, J = 7.8, 1 H, H4)
12.0)
[73.12]
overlap-
ping
[85.38]
[104.1 (s, 3JC4,F6 < 1)], [125.57 (d, 3J7a,F6 = 12.51,
C7a), 139.86 (s, C4a), 143.30 (d, 2JC5,F6 = 13.12,
C5)], 5.27 (br s, 3 OH), 4.12 (q, 2 H, CH2CH3)
[64.53], 1.36 (t, J = 6.9 Hz, 3 H, CH2CH3) [14.24]
OCH2CH3
m)
[69.85]
[60.94]
14b
15a
5.84 (d, J = 4.36 (dd)
–
4.14 (m)
[69.85]
3.99 (m)
[85.38]
3.61–3.69 8.32 (s, 1 H, H2) [142.5], 7.62 (d, J = 7.8, 1 H, H7)
(m)
[60.94]
6.4)
[73.12]
[97.31 (s, 3JC7,F5 < 1)], 7.49 (d, J = 11.6, 1 H, H4)
[105.5 (d, 3JC7,F5 = 20.96)], 4.13 (q, 2 H, CH2CH3)
[64.53], 1.38 (t, 3 H, CH2CH3) [14.0]
[88.64]
6.28 (dd,
2.54 (m) 2.26 (m)
4.38 (m)
[70.93]
3.86 (m)
[87.95]
3.57 (dd, 8.37 (s, 1 H, H2) [142.78], 7.68 (d, 3JH7,F6 = 11.4, 1
dd, J =
H, H7) [99.49 (d, 2JC7,F6= 25.2)], 7.37 (d, 4JH4,F6
J = 7.5, 6.2) [40.10]
[85.30]
=
3.8, 4.1, 7.9, 1 H, H4) [105.10 (s, 3JC4,F6 < 1)], 5.05 (br s, 3
gemJ =
11.7)
OH), 8.37 (s, 1 H, H2) [142.78], 4.11 (m ,2 H,
CH2CH3) [65.22], 1.36 (t, 3 H, CH2CH3), [15.08]
[61.93]
15b
16a
6.34 (dd,
J = 7.7, 6.1) [40.10]
[85.23]
2.56 (m) 2.26 (m)
4.41 (m)
[70.98]
3.88 (m)
[87.975]
3.59 (dd, 8.34 (s, 1 H, H2) [142.44], 7.56 (d, 3JH7,F5 = 7.9, 1 H,
dd)
[61.93]
H7) [97.62 (s, 3JC7,F5 < 1.0, C7)], 7.47 (d, 4JH4,F5
=
11.6, 1 H, H4) [106.07 (d, 2JC4,F5 = 20.96, C4)], 5.30
(br s, 3 OH), 8.34 (s, 1 H, H2) [142.44], 4.13 (m, 2
H, CH2CH3) [65.22], 1.37 (t, 3 H, CH2CH3) [15.08]
5.79 (d, J = 4.31 (m)
6.5)
[88.63]
–
4.10 (m)
[69.78]
3.97 (m)
[85.29]
3.64, 3.66 8.36 (s, 1 H, H2) [142.74], 7.73 (d, 3JH7,F6 = 12.4, 1
[73.05]
(m)
[60.97]
H, H7) [99.06], 7.40 (d, 4JH4,F6 = 7.9, 1 H, H4) [107.
37], 5.41 (br s, 2′-OH); 5.20 (br s, 3′-OH), 5.19 (br
s, 5′-OH), 4.58 (m, 1 H, CHMe2) [71.95],
1.29 (d, 6 H, 2 CH3) [21.66]
Synthesis 2013, 45, 272–280
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279
Table 5 Selected 1H and 13C NMR Spectroscopy Data of Benzimidazole Nucleosides (DMSO-d6) δ, J (Hz)a (continued)
Compd H1′
[C1′]
H2′a
[C2′]
H2′b
[C2′]
H3′
[C3′]
H4′
[C4′]
H5′a/H5′b Other [13C]
[C5′]
17ac
6.28 (dd,
J = 7.6, 6.1) [38.26]
[85.24]
2.55 (m) 2.26 (octet, 4.39 (m)
3.82 (m)
[87.91]
3.52 (m) 8.34 (s, 1 H, H2) [143.04], 7.68 (d, 3JH7,F6 = 11.2, 1
J = 13.3,
[70.17]
[61.17]
H, H7) [99.61 (d, 2JC7,F6 = 24.0)], 7.36 (d, 4JH4,F6
=
6.0, 3.1)
7.8, 1 H, H4) [107.99 (s, 3JC4,F6 < 1)], 5.33 (d, J =
3.8, 3′-OH), 5.01 (t, J = 5.1, 5′-OH), 4.58 (m, 1 H,
CHCH3) [72.56], 1.28 (d, J = 6, 6 H, CHCH3)
[21.56]
17bd
18a
6.31 (dd,
~2.55 (m) ~2.26 (m) ~4.39 (m) ~3.83 (m)
~3.52 (m) 8.31 (s, 1 H, H2) [142.55], 7.54 (d, 4JH7,F5 = 7.8, 1 H,
J = 7.8, 5.9) [–b]
[–b]
[–b]
[–b]
[–b]
H7) [100.03], 7.44 (d, 3JH4,F5 = 11.6, 1 H, H4)
[106.18], 5.33 (br s, 3′-OH), 5.02 (br s, 5′-OH), 4.64
(m, 1 H, CHCH3) [72.33], 1.30 (d, 6 H, CHCH3)
[85.26]
5.79 (d, J = 4.30 (m)
6.5)
[88.64]
–
4.10 (m)
[69.79]
3.97 (m)
[85.26]
3.65 (m) 8.37 (s, 1 H, H2) [142.59], 7.70 (d, 3JH7,F6 = 12.4, 1
[73.10]
[60.99]
H, H7) [99.19 (d, 2JC7,F6 = 27.9)], 7.31 (d, 4JH4,F6
=
7.9, 1 H, H4) [108.92 (s, 3JC4,F6 < 1)], 5.40 (br s, 2′-
OH), 5.18 (br s, 3′-OH), 5.15 (br s, 5′-OH), 3.76 (m,
4 H, O(CH2)2) [60.26], 2.98 (m, 4 H, NCH2) [51.27]
19a
20a
6.28 (dd,
J = 7.1, 6.4) [39.24]
[84.41]
2.54 (m) 2.25 (m)
4.38 (m)
[70.05]
3.86 (m)
[87.07]
3.55, 3.58 8.37 (s, 1 H, H2) [142.07], 7.64 (d, 3JH7,F6 = 12.4, 1
(dd, dd, H, H7) [98.84 (d, 2JC7,F6 = 27.72)], 7.30 (d, 4JH4,F6
gemJ = 7.9, 1 H, H4) [108.83 (s, 3JC4,F6 < 1)], 5.31 (br s, 3′-
11.7, 4.0) OH), 4.98 (br s, 5′-OH), 3.76 (m, 4 H, OCH2CH2)
[61.08] [65.97], 2.97 (m, 4 H, NCH2CH2) [51.16]
=
5.78 (d, J = 4.30 (m)
6.3)
[88.62]
–
4.10 (m)
[69.78]
3.97 (m)
[85.20]
3.6–3.7 8.34 (s, 1 H, H2) [142.47], 7.66 (d, 3JH7,F6 = 12.4, 1
[73.06]
(m)
[60.93]
H, H7) [99.03], 7.29 (d, 4JH4,F6 = 7.9, 1 H, H4)
[108.97], 5.38 (d, J = 6.5, 1 H, 2′-OH), 5.15 (d, J =
4.7, 1 H, 3′-OH), 5.12 (t, J = 5.0, 1 H, 5′-OH), 2.98
(m, 4 H, MeNCH2CH2) [50.73], 2.50 (m, 4 H,
MeNCH2CH2) [54.59], 2.24 (s, 3 H, NCH3) [45.51]
21a
6.28 (dd,
J = 7.3, 6.4) [39.14]
[84.60]
2.54 (m) 2.26 (m)
4.38 (m)
[70.16]
3.86 (m)
[87.37]
3.51–3.61 8.36 (s, 1 H, H2) [142.30], 7.61 (d, 3JH7,F6 = 12.4, 1
(m)
[61.21]
[39.14]
H, H7) [98.97], 7.28 (d, 4JH4,F6 = 7.9, 1 H, H4)
[109.12], 5.29 (d, J = 4.1, 1 H, 3′-OH), 4.96 (t, J =
5.2, 1 H, 5′-OH), 2.50 (m, 4 H, MeNCH2CH2)
[54.50], 2.98 (m, 4 H, MeNCH2CH2) [50.79], 2.24
(s, 3 H, NCH3) [45.48]
a 1H, 13C, and 15N resonances were assigned using 2D homo- and heteronuclear experiments: 1H/1H-COSY, 1H/13C-HSQC, 1H/13C-HMBC,
1H/15N-HSQC, 1H/15N-HMBC, and [1H,1H] NOE spectra.
b Not determined, for example, the quantity of isomer 17b (4%) in the mixture with 17a was not sufficient to determine its 13C nuclear
shifts in 1H/13C-HSQC, 1H/13C-HMBC spectra.
c Mixture: 96%.
d Mixture: 4%.
G. W.; van Draanen, N.; Ptak, R. G.; Drach, J. C.;
Acknowledgment
Townsend, L. B. J. Med. Chem. 2000, 43, 2464. (d) Sharma,
Financial support by the International Science and Technology
D.; Narasimhan, B.; Kumar, P.; Judge, V.; Narang, R.; De
Centre (project #B-1640), the Ministry of Education and Science of
Russian Federation, State Contract 02.740.11.0260, and the KU
Leuven (GOA 10/14) are gratefully acknowledged. The authors are
Clercq, E.; Balzarini, J. J. Enzyme Inhib. Med. Chem. 2009,
24, 1161.
(3) (a) Prichard, M. N. Rev. Med. Virol. 2009, 19, 215.
indebted to Dr. Roman S. Esipov (Shemyakin-Ovchinnikov Insti-
(b) Williams, S. L.; Hartline, C. B.; Kushner, N. L.; Harden,
tute of Bioorganic Chemistry, Moscow) for supplying the recombi-
nant E. coli nucleoside phosphorylases.
E. A.; Bidanset, D. J.; Drach, J. C.; Townsend, L. B.
Antimicrob. Agents Chemother. 2003, 47, 2186. (c) Hwang,
J. S.; Kregler, O.; Schilf, R.; Bannert, N.; Drach, J. C.;
Townsend, L. B.; Bogner, E. J. Virol. 2007, 81, 11604.
(d) Hwang, J. S.; Schilf, R.; Drach, J. C.; Townsend, L. B.;
References
Bogner, E. Antimicrob. Agents Chemother. 2009, 53, 5095.
(4) For reviews, see: (a) Trofe, J.; Pote, L.; Wade, E.; Blumberg,
E.; Bloom, R. D. Ann. Pharmacother. 2008, 42, 1447.
(b) Drugs R D; 2007, 8; 188 Maribavir: 1263W94,
(1) For a review, see: De Clercq, E. Adv. Virus Res. 2009, 73, 1.
(2) (a) Krosky, P. M.; Borysko, K. Z.; Nassiri, M. R.; Devivar,
R. V.; Ptak, R. G.; Davis, M. G.; Biron, K. K.; Townsend, L.
B.; Drach, J. C. Antimicrob. Agents Chemother. 2002, 46,
478. (b) Evers, D. L.; Komazin, G.; Shin, D.; Hwang, D. D.;
Townsend, L. B.; Drach, J. C. Antiviral Res. 2002, 56, 61.
(c) Gudmundsson, K. S.; Tidwell, J.; Lippa, N.; Koszalka,
Benzimidavir, GW 1263, GW 1263W94, VP41263.
(c) Chou, S.; Marousek, G. I. J. Virol. 2008, 82, 246.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 272–280

280
I. D. Konstantinova et al.
PAPER
(12) Kotovskaya, S. K.; Perova, N. M.; Baskakova, Z. M.;
Romanova, S. A.; Charushin, V. N.; Chupakhin, O. N. Russ.
J. Org. Chem. 2001, 37, 564.
(13) Kotovskaya, S. K.; Charushin, V. N.; Chupakhin, O. N.;
Kozhevnikova, E. O. Russ. J. Org. Chem. 1998, 34, 369;
Chem. Abstr. 1999, 130, 25028.
(5) For reviews, see: (a) Galmarini, C. M.; Popowycz, F.;
Joseph, B. Curr. Med. Chem. 2008, 15, 1072. (b) Mathe, C.;
Gosselin, G. Antiviral Res. 2006, 71, 276. (c) Biron, K. K.
Antiviral Res. 2006, 71, 154.
(6) Mikhailopulo, I. A. Curr. Org. Chem. 2007, 11, 317.
(7) Mikhailopulo, I. A.; Zinchenko, A. I.; Bokut, S. B.; Dudchik,
N. V.; Barai, V. N.; Kalinichenko, E. N.; Rosemeyer, H.;
Seela, F. Biotechnol. Lett. 1992, 14, 885.
(8) Mikhailopulo, I. A.; Kazimierczuk, Z.; Zinchenko, A. I.;
Barai, V. N.; Romanova, V. V.; Eroshevskaya, L. A. Nucl.
Acids Symp. Ser. 1994, 31, 83.
(9) Mikhailopulo, I. A.; Kazimierczuk, Z.; Zinchenko, A. I.;
Barai, V. N.; Romanova, V. V.; Eroshevskaya, L. A.
Nucleosides Nucleotides 1995, 14, 477.
(14) Berger, S.; Braun, S.; Kalinowski, H.-O. 19F NMR-
Spektroskopie, In NMR Spektroskopie von Nichtmetallen;
Vol. 4; Georg Thieme Verlag: Stuttgart, 1994.
(15) Roivainen, J.; Elizarova, T.; Lapinjoki, S.; Mikhailopulo, I.
A.; Esipov, R. S.; Miroshnikov, A. I. Nucleosides,
Nucleotides Nucleic Acids 2007, 26, 905.
(16) Kulikovskiy, A. I.; Esipov, R. S.; Miroshnikov, A. I.;
Mikhailopulo, I. A. Collect. Symp. Ser. 2011, 12, 397.
(17) Konstantinova, I. D.; Antonov, K. V.; Fateev, I. V.;
Miroshnikov, A. I.; Stepchenko, V. A.; Baranovsky, A. V.;
Mikhailopulo, I. A. Synthesis 2011, 1555.
(10) Bentancor, L.; Trelles, J. A.; Nóbile, M.; Lewkowicz, E. S.;
Iribarren, A. M. J. Mol. Catal. B: Enzym. 2004, 29, 3.
(11) Esipov, R. S.; Gurevich, A. I.; Chuvikovsky, D. V.;
Chupova, L. A.; Muravyova, T. I.; Miroshnikov, A. I.
Protein Expression Purif. 2002, 24, 56.
(18) Mikhailopulo, I. A.; Miroshnikov, A. I. Acta Naturae 2010,
2, 36.
Synthesis 2013, 45, 272–280
© Georg Thieme Verlag Stuttgart · New York