Chemoenzymatic Synthesis and Antiherpes Activity of 5-Substituted 4,6-Difluorobenzimidazoles Ribo- and 2′-Deoxyribonucleosides

Article abstract of DOI:10.1055/s-0035-1560911

A series of 5,6-disubstituted benzimidazole nucleosides, obtained earlier, did not show any significant antiviral activity at relatively low cytotoxicity in vitro. In the course of our research we have succeeded in introducing an additional fluorine atom

Full text of DOI:10.1055/s-0035-1560911

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
©
Georg Thieme Verlag Stuttgart · New York
2016, 48, 394–406
394
paper
Synthesis
M. I. Kharitonova et al.
Paper
Chemoenzymatic Synthesis and Antiherpes Activity of 5-Substi-
tuted 4,6-Difluorobenzimidazoles Ribo- and 2′-Deoxyribonucleo-
sides
Maria I. Kharitonova*a
Ilja V. Fateeva
Alexei L. Kayushina
Irina D. Konstantinovaa
Svetlana K. Kotovskayab,c
O
N
X
HN
O
O
P
HO
HO
UP, Pi
– Ura
O
O
F
–OH
O
O
R
HO
X
N
N
Valeria L. Andronovad
Georgii A. Galegovd
HO
Urd, dUrd
PNP
Pi
F
Valery N. Charushinb,c
Anatoly I. Miroshnikova
–
HO
F
F
O
Y
NH2
R
2
or 3 steps
N
HO
X
a
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
F
NO2
Y = H, F
F
N
Russian Academy of Sciences, Miklukho-Maklaya St. 16/10,
H
mixture of N-1 (prevalent), N-3 (minor)
isomers, chromatography-free
117997, Moscow, Russian Federation
R = H, F, OMe, OEt,
morpholino, pyrrolidino
12 examples 60–90% yield
kharitonova-mari@rambler.ru
b
c
Postovsky Institute of Organic Synthesis, the Ural Branch of
the Russian Academy of Sciences, S.Kovalevskaya/Academich-
eskaya St. 22/20, 620041, Ekaterinburg, Russian Federation
Ural Federal University named after the first President of Rus-
sia B.N. Yeltsin, Mira St. 19, 620002, Ekaterinburg, Russian
Federation
Ivanovsky Research Institute of Virology, Russian Academy of
Medical Sciences, Gamalei St. 16, Moscow, 123098, Russian
Federation
d
Received: 10.07.2015
Accepted after revision: 09.10.2015
Published online: 23.11.2015
bine, and their analogues, in medical practice.1,2 Derivatives
of benzimidazole (BzIm), which exhibit high antiviral activ-
ity (such as purine and pyrimidine nucleosides), are of spe-
cial interest in this respect.3 The most effective com-
pounds of the class β-L- and β-D-BzIm-nucleosides include
maribavir (1) [2-isopropylamino-5,6-dichloro-1-(β-L-ribo-
furanosyl)benzimidazole], 5,6-dichloro-1-(β-L-ribofurano-
syl)benzimidazole (2), 2,5,6-trichloro-1-(β-D-ribofurano-
syl)benzimidazole (3), and 2-bromo-5,6-dichloro-1-(β-D-
ribofuranosyl)benzimidazole (4) (Figure 1). These com-
pounds effectively suppress replication of cytomegalovirus
DOI: 10.1055/s-0035-1560911; Art ID: ss-2015-t0386-op
–5
Abstract A series of 5,6-disubstituted benzimidazole nucleosides, ob-
tained earlier, did not show any significant antiviral activity at relatively
low cytotoxicity in vitro. In the course of our research we have succeed-
ed in introducing an additional fluorine atom into the benzimidazole
ring system. A new series of 4,6-difluorobenzimidazoles, bearing vari-
ous groups (fluoro-, methoxy-, ethoxy-, morpholino-, and pyrrolidino-)
in the 5-position of the benzene ring, have been synthesized. All these
compounds proved to be substrates for recombinant E. coli purine nu-
cleoside phosphorylase (PNP) in the transglycosylation reaction. Effec-
tive methods for the synthesis of ribo- and 2′-deoxyribonucleosides
with high yields (60–90%) have been described, and the formation of
regioisomeric N3-nucleosides of benzimidazoles have been detected.
The biological activity of the nucleosides obtained against herpes sim-
plex virus type 1 (HSV-1) has been elucidated. All compounds show a
low cytotoxicity in the cell culture Vero E6. 4,5,6-Trifluoro-1-(β-D-ribo-
furanosyl)benzimidazole and 5-methoxy-4,6-difluoro-1-(β-D-2′-deoxy-
ribofuranosyl)benzimidazole proved to inhibit completely the progres-
sion of the virus cytopathic effect (CPE) at a multiplicity of infection
(
CMV), human herpes simplex viruses types 1 and 2 (HSV-1
6–8
and HSV-2), and herpes virus type 6.
Currently, clinical
trials of Maribavir (1), as a drug for prevention and treat-
ment of CMV infections after transplantation of allogeneic
bone marrow and parenchymatous organs are expected to
be completed.9 Maribavir has just achieved the status of
an orphan drug for CMV infections in the USA.13
–12
In the course of previous studies it was shown that com-
pounds 5 and 6 were inactive against a wide spectrum of
(MOI) of 0.01 PFU/cell.
14,15
DNA and RNA viruses,
and nucleosides 6 did not exhibit
Key words drug discovery, herpes simplex virus, fluorine, nucleosides
a high cytotoxicity.14 However, it is well known that incor-
poration of a fluorine atom into the heterocyclic base of nu-
cleosides can lead to a change in their properties, including
their biological activity (for instance, anticancer drug
Fludarabine is a C2-fluoro analogue of the antiviral drug
Vidarabine).16 We have introduced a fluorine atom into the
The development of new drugs based on modified nu-
cleosides has attracted the continuing interest of chemists
and biologists because of the well-established value of
drugs such as Acyclovir, Azidothymidine, Ribavirin, Clofara-
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M. I. Kharitonova et al.
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R1
R1
R1
R1
N
N
Cl
Cl
Cl
N
N
N
N
N
R2
R
R
N
F
Cl
R1
O
HO
HO
HO
HO
O
O
O
R2
OH
OH
OH
OH
OH
OH
5a, R1 = Br, R2 = H
5b, R1 = H, R2 = Br
6, R1 = F, OMe, OEt, Oi-Pr, etc.
R2 = OH, H
1
2
, R = NHi-Pr
, R = H
3, R = Cl
4, R = Br
Figure 1 Benzimidazole nucleosides
C4-position of benzimidazole, and have obtained the corre-
sponding ribo- and 2′-deoxyribonucleosides to study the
antiviral activity of these compounds. The synthesis of the
nucleosides was performed by enzymatic transglycosyla-
tion reaction catalyzed by recombinant nucleoside phos-
We have suggested a new approach for reduction of ni-
troanilines 7 and 8 into the corresponding amines 9 and 10
by using tin in hydrochloric acid, followed by cyclization in
formic acid (Scheme 1). Notably, this method does not re-
quire chromatographic purification of 11 or 12.
17–20
phorylases E. сoli.
The fluorine atom in the para-position relative to the ni-
tro group in 8 can be easily displaced with nucleophiles. In-
deed, a number of 5-substituted 2,4-difluoro-6-nitroani-
lines (13a–d) have been synthesized by using the reaction
of 8 with alcoholates (OMe, OEt) and amines (morpholine,
pyrrolidine). Compounds 13 were transformed into the cor-
responding diamino compounds 14 through catalytic re-
duction or through the use the Sn/HCl system (Scheme 2).
The best results were obtained when reduction was
performed by heating a solution of nitroaniline 13a or 13b
to reflux in the corresponding alcohol with hydrazine hy-
drate in the presence of Raney Ni (1 h, 70–80% yield). Ni-
troanilines 13c and 13d were reduced to ortho-phenylene-
diamines 14c and 14d in 40–70% yields, by using tin in hy-
The synthesis of 4,6-difluorobenzimidazole (11) from
,4-difluoro-6-nitroaniline (7) was described previous-
2
21,22
ly.
ortho-Phenylenediamine 9 was obtained by reduc-
tion of 7 with H in the presence of platinum dioxide. 4,6-
2
Difluorobenzimidazole (11) was synthesized by the reac-
tion of 9 with diethoxymethyl acetate (total yield 28%). The
synthesis of 4,5,6-trifluorobenzimidazole (12) from 2,3,4-
trifluoro-6-nitroaniline has also been described previous-
23–26
ly.
The nitro group was reduced with H in the presence
2
of Raney nickel to give 10. The imidazole ring was formed
through cyclization, which proceeded smoothly in formic
acid heated to reflux; compound 12 was purified by chro-
matography.
drochloric
acid.
5-Substituted
4,6-difluoro-1H-
benzimidazoles were synthesized by condensation of 14a–
d in formic acid heated to reflux (40–60% yield). A selection
of physicochemical properties for compounds 11, 12, and
15–18 are given in Table 1.
The transglycosylation reaction conditions for the syn-
thesis of β-D-ribo- and β-D-2′-deoxyribonucleosides of the
family of 4,5,6-trisubstituted benzimidazoles (Table 2) were
optimized. Thus, an appropriate donor of ribofuranose or
deoxyribofuranose was chosen, the optimal ratio between
F
F
F
X
F
NH2
X
F
NH2
NH2
X
F
N
Sn/HCl
HCOOH
O
N
H
N
O
7
, 8
9, 10
11, 12
7
8
, 9, 11, X = H
, 10, 12, X = F
Scheme 1 Synthesis of 4,6-difluoro- and 4,5,6-trifluoro-1H-benzimid-
azoles
F
F
F
N2H4
NI-Ra
Y
F
NH2
NO2
Y
NH2
NH2
Y
F
YH
HCOOH
N
8
or Sn/HCl
N
H
F
13a–d
14a–d
15–18
Y = OMe (13a, 14a, 15); OEt (13b, 14b, 16);
N
O (13c, 14c, 17);
N
(13d, 14d, 18)
Scheme 2 Synthesis of 5-substituted 4,6-difluoro-1H-benzimidazoles
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1
Table 1 Physicochemical Properties and H NMR Data for 11, 12, and 15–18
Yield (%)a Mp (°C) , appearance
b
R
c
Purity (%) 1H NMR (DMSO-d
[t , min]
R
), δ (ppm), J (Hz)
Compound
f
6
d
H-2 (s)
8.10
H-7
NH (br s) Other
12.66 7.12 (d, J = 8.0, 1 H, H-5),
12.75
1
1
78
56
40
45
60
50
≥ 190
light-brown crystals
0.57
0.65
0.65
0.66
0.63
0.66
99.4
[5.8]
6.79 (m)
7.31 (m)
(X = H)
1
2
230–232
colorless crystals
98.4
[7.2]
8.15
8.54
8.05
8.03
8.05
–
(X = F)
1
5
193–195
light-brown crystals
99.2
[6.9]
7.64 (d, J =
9.5)
13.04
12.54
12.60
12.50
3
4.38 (s, 3 H, OCH )
(
Y = OMe)
1
6
158–160
cream crystals
96.6
[8.9]
7.18 (d, J =
9.6)
1.37 (t, J = 7.0, 3 H, OCH
(dd, J = 6.9, 2 H, OCH CH )
2
CH
3
3
), 4.10
(
Y = OEt)
2
1
7
180–182
light-brown crystals
96.7
[7.9]
7.10 (d, J =
10.8)
2 2
3.12 (t, J = 4.3, 4 H, CH NCH
), 3.73
(
Y = morpholino)
2
2
(t, J = 4.5, 4 H, CH OCH )
1
8
183–185
light-brown crystals
96.5
[8.6]
7.08 (d, J =
11.3)
1.94 (m, 4 H, CH NCH ), 3.33 (m,
2
2
(
Y = pyrrolidino)
2
2
4 H, CH CH )
a
b
c
Isolated yield: satisfactory C, H, and N (±0.40) elemental analyses were obtained.
Solvent: H O–CH CN, 1:1.
TLC: Sorbophil (Merck); CHCl
2
3
3
–EtOH, 10:1
d
HPLC (Waters 1525, Waters 2487, Breeze 2; column: Nova Pack C18, 4 μm, 4.6 × 150 mm; eluent А: 0.1% ТFA–H
2 2
O, eluent В: 70% MeCN in 0.1% ТFA–H O; flow:
1
mL/min; UV detection: 254 and 280 nm); elution: 0 to 70% В over 20 min.
heterocyclic base and sugar moiety was determined, and
the required quantity of enzymes was found.
The natural ribonucleosides (uridine, inosine, and gua-
nosine) and their 2′-deoxy analogues have been used as do-
nors of a sugar residue. When transglycosylation takes
place between purine nucleoside and BzIm, only purine nu-
cleoside phosphorylase (PNP) E.coli was used. When trans-
glycosylation was performed between uridine (Urd)/deoxy-
uridine (dUrd) and BzIm, both uridine phosphorylase (UP)
and PNP were required. The reactions proceeded at 50 °C in
Table 2 Enzymatic Synthesis of 5-Substituted 4,6-Difluorobenzimidazole Ribo- and 2′-Deoxyribonucleosides 19–30a
O
F
HN
R
N
N
F
N
N
base Bzlm
PNP
O
N
X
O
P
HO
HO
F
R
UP, Pi
Ura
O
O
OH
+
F
O
–
– Pi
HO
HO
O
O
HO
HO
X
O
Urd/dUrd
X = OH, H
HO
X
HO
X
Compd
X
R
Compd
X
R
1
2
9
0
OH
H
25
26
OH
H
OEt
H
2
2
2
1
2
3
4
OH
H
F
27
28
29
30
OH
H
N
O
OH
OMe
OH
N
2
H
H
a
Definitions: Pi = inorganic phosphate; UP = uridine nucleoside phosphorylase; PNP = purine nucleoside phosphorylase; Urd = uridine; dUrd = deoxyuridine.
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potassium dihydrogen phosphate buffer (4–10 mM, pH 7),
and the progress of the reaction was monitored by reverse-
phase HPLC.
Conversion of a heterocyclic base into nucleoside
proved to be higher when Urd was used as ribose donor
equilibrium conversion is observed in all reactions, and it
does not depend on the quantity of added NPs. So, for pre-
parative synthesis of BzIm-ribosides, we decreased the
PNP/UP ratio to 45 e.u./mmol base PNP and 30 e.u./mmol
Urd or dUrd (Table 3). This increased the total reaction time,
but reduced the amount of enzyme required per run.
(Figure 2). The optimal nucleoside/base ratio was 2:1. An
increase in the ratio gave no significant changes in the level
of conversion (Figure 3).
Figure 2 Dependence of base 12 conversion into riboside 21 on the
type of ribofuranose donor. Reagents and conditions: base (1 mM), sugar
moiety donor (2 mM), buffer (pH 7.0, 2 mM KH PO , 1.0 mL), recombi-
2
4
nant E. coli enzymes [1800 e.u. of PNP and 1800 e.u. of UP (only for
Urd)], 50 °C, 24 h.
Figure 4 Dependence of base 11 conversion into (a) riboside 19 and
(
b) deoxyriboside 20 on NPs ratio. Reagents and conditions: base (1
mM), sugar moiety donor (1.5 mM), buffer (pH 7.0, 1.5 mM KH PO ,
2
4
0.5 mL), X e.u. E. coli of PNP and Y e.u. of UP, 50 °C, 168 h (a) or 2 h (b).
X (e.u./mmol base)/ Y (e.u./mmol Urd or dUrd) ratios: 1–900/600;
2–360/240; 3–360/60; 4–90/240; 5–90/60.
In the case of 2′-deoxyriboside, this synthetic procedure
was not optimal; here, the conversion decreased (10–15%)
after reaching a maximum in the first two hours (Figure 4,
b). This phenomenon can be explained by hydrolysis of 2-
deoxyribose-α-phosphate with participation of the active
Figure 3 Dependence of base 18 conversion into riboside 29 on the
Urd/base 18 ratio. Reagents and conditions: base (1 mM), sugar moiety
donor (X mM), buffer (pH 7.0; X mM KH PO , 1.0 mL), 1800 e.u. of E.
2
4
coli PNP and 1800 e.u. of UP, 50 °C, 12 h. X = 1, 1.5, 2, 3, or 5 mM.
site of the enzyme into inorganic phosphate and 2-deoxyri-
bose.2
7–29
The 2-deoxyribose is not substrate for NPs in the
transglycosylation reaction, and the equilibrium of trans-
glycosylation reaction is displaced to the formation of the
original base. Therefore, the synthesis of 2′-deoxyribonu-
cleosides were not allowed to proceed for more than 3–4
hours.
To establish a dependence of the conversion of bases
into nucleosides we used 90–900 e.u. PNP per 1 mmol het-
erocyclic base and 60–600 e.u. UP per 1 mmol of either Urd
or dUrd.
As can be seen from Figure 4, during the initial hours an
increase in the amount of enzyme accelerates the formation
of the target products. However, after a prolonged time, the
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Table 3 Experimental Data for the Enzymatic Synthesis of Benzimidazole β-D-Ribo- and β-D-2′-Deoxyribonucleosides 19–30a
Compd,
mol wt
Acceptor,
mol wt
Donor,
mol wt
Volume (mL)
Substrates
Acceptor
PNP
UP
Time
Conv. base→ Eluent
(e.u.) [A]b
(e.u.) [B]b (h)
nucleoside (%)c (%)
d
[mM KН
2
PO
4
]
Donor
Ratio
1:2
(
mg) [mmol] (mg) [mmol] A/D
1
9
11
154.11
Urd
244.2
114
[8]
70
[0.46]
223
[0.91]
55
[120]
27
[30]
72
95.1
86.1
98.7
98.7
92.3
90.5
96.9
88.2
93.0
94.3
83.8
98.7
20
25
–
286.23
2
0
dUrd
228.2
114
[5]
70
[0.41]
156
[0.61]
1:1.5
82
[200]
73
[120]
3
192
3
270.23
2
1
12
172.09
Urd
102
[8]
70
[0.41]
199
[0.82]
1:2
98
[240]
98
[120]
304.22
2
2
dUrd
Urd
102
[8]
70
[0.41]
186
[0.82]
1:2
185
[450]
197
[240]
–
288.22
2
3
15
184.14
95
[8]
70
[0.38]
186
[0.76]
1:2
17
[45]
23
[30]
24
3
25
20
30
22
50
40
50
40
316.26
2
4
dUrd
Urd
95
[6]
70
[0.38]
130
[0.57]
1:1.5
1:1.5
1:1.3
1:2
171
[450]
171
[300]
300.26
2
5
16
198.17
88
[6]
70
[0.35]
129
[0.53]
10
[30]
8
[15]
24
4
330.29
2
6
dUrd
Urd
88
[5]
70
[0.35]
100
[0.44]
158
[450]
158
[360]
314.29
2
7
17
239.21
73
[8]
70
[0.29]
143
[0.58]
13
[45]
17
[30]
48
371.34
2
8
dUrd
Urd
68
[10]
65 мг
[0.27]
155
[0.68]
1:2.5
1:2
122
[450]
122
[180]
2.5
355.34
2
9
18
223.21
157
[4]
70
[0.31]
152
[0.63]
14
[45]
19
[30]
48
2
355.34
3
0
dUrd
157
[4]
70
[0.31]
143
[0.63]
1:2
14
[45]
113
[180]
339.34
a
All enzymatic reactions were performed in potassium phosphate buffer (4–10 mM; pH 7.0) at 50 °C and the progress of the reaction was monitored by HPLC
analysis. The reactions were stopped by addition of ethanol, when base was transformed into nucleoside as observed in test reactions. The reaction mixture was
then evaporated to 5 mL volume. Compounds 21 and 22 were crystallized from water; the other products were purified by silica gel C18 (Merck) column (20 × 100
mm) chromatography.
b
Definitions: A = ratio of PNP (enzyme units)30 per base (1 mmol); B = ratio of UP (enzyme units)30 per nucleoside [uridine (Urd) or 2′-deoxyuridine (dUrd)] (1
mmol).
c
Based on HPLC analysis.
Nucleosides were eluted from the C18 column with 1) H
d
2 2 2
O–MeCN for 19, 20, and 23–26; 2) H O–EtOH (1:1) for 27 and 29; 3) H O–40% MeOH for 28 and 30.
Eluent: % (vol) MeCN or alcohol.
According to previous reports, the optimal pH value of
20
the reaction is 6–8. The conversion of benzimidazole nu-
cleosides does not depend on pH in this interval (Figure 5).
Notably, transglycosylation proceeds even at pH 4.0 and
10.0, albeit rather slowly.
Similar to the synthesis of nucleosides from disubstitut-
ed benzimidazoles,1 a mixture of regioisomeric N1- (a) and
N3-nucleosides of benzimidazoles (b) was obtained from
the transribo- and 2′-deoxyribosylation reactions of the
corresponding heterocyclic bases (compounds 19–30; Table
4
4). However, in the case of ribo- and 2′-deoxyribonucleo-
sides of morpholino (27, 28) and pyrrolidino (29) deriva-
tives, the formation of N3-regioisomers was not detected by
LC/MS and NMR analysis.
The level of conversion for the transformation of 13–18
into nucleosides was 84–99%. Compounds 21 and 22 were
isolated by crystallization; all other compounds were iso-
Figure 5 Dependence of base 11 conversion into riboside 19 on pH.
Reagents and conditions: base (1 mM), sugar moiety donor (2 mM),
buffer (pH 7.0; 2 mM KH
PO , 1.0 mL), 1800 e.u. of E. coli PNP and
2
4
1800 e.u. of UP, 50 °C, 96 h.
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Table 4 Conversion of Benzimidazoles 11–12 and 15–18 into Nucleo-
sides 19–30, and the Ratio of Isomers N1 (a) and N3 (b)
Physicochemical properties and 1H and 13C NMR data
including [ H, H] and [ H, C] 2D: COSY, HSQC, HMBC and
1
1
1
13
(
NOE) for all regioisomers of the synthesized nucleosides are
presented in the experimental section.
Compound
Conv. (%)
Ratio of isomersa
a
b
Cytotoxic properties of compounds 19–30 relative to
Vero E6 cells were determined by incubating a cell culture
in the presence of benzimidazole nucleosides at the maxi-
mal investigated concentration (1000 μg/mL) during 72
hours (Table 5). Compounds 23, 27, 28, and 29 did not ex-
hibit a cytotoxicity within the concentrations studied. Oth-
er compounds exhibited cytotoxicity under these condi-
tions, as detected by the presence of dead cells in the exper-
imental system (the number of dead cells in experimental
system was more than the number of dead cells in control).
In this group of compounds, nucleosides 24 and 26 had the
least cytotoxicity (CD50 = 745 and 742.5 μg/mL, respective-
1
2
2
2
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
8
9
0
95.2
86.1
98.6
98.7
92.3
90.5
96.9
88.2
93.0
94.3
83.8
98.7
87
88
13
12
10
8
90
92
91
9
94
6
91
9
90
10
nd
nd
nd
11
100
100
100
89
ly). Compound 30 proved to be the most toxic (CD50
12.5 μg/mL).
Antiviral activity of the compounds against a herpes
=
5
3
simplex virus type 1 (HSV-1) model was then investigated.
Compounds 23, 27, and 28 did not exhibit an antiviral effect
up to a concentration 1000 μg/mL. Compounds 20 and 22
also did not inhibit the progression of virus-induced cyto-
pathic effect (CPE) in a concentration range that was not
toxic for the cell culture. Nucleosides 19, 25, 26, and 29
showed minimal antiviral activity when used in subtoxic
concentrations at a low multiplicity of infection (MOI) only
(0.01 PFU/cell). Compound 30 exhibited minimal activity at
a
Based on HPLC analysis; nd = not detected by HPLC.
lated by reverse-phase column chromatography. The syn-
thesis of 2′-deoxyribosides was usually completed in 0.5–
.0 hours. The transribosylation proceeded much more
slowly, and was completed in 1–8 days. General experimen-
tal data for the enzymatic synthesis of β-D-ribo- and β-D-2′-
deoxyribonucleosides are shown in Table 3.
3
Table 5 In Vitro Antiviral Activity of 19–30 Against HSV-1
Compd
Max. tolerable concn CD50 (μg/mL)b
ID50 (μg/mL)c
ID95 (μg/mL)d
IS (CD50/ID50
0.1
)
a
(MTC)
MOI (PFU/mL)e
0.1
0.01
125
0.1
0.01
500
0.01
Ribavirin
>500
>500
250
>500
>500
>2
0
>4
1.5
1
2
2
2
2
2
2
2
2
2
2
3
9
0
1
2
3
4
5
6
7
8
9
0
500
500
735 ± 10
707.5 ± 2.5
542.5 ± 52.5
662.5 ± 17.5
>1000
>500
>500
250
500
>500
>500
500
>500
>500
0
0
125
62.5
>500
2
8.68
0
>500
>1000
500
>500
>1000
500
>500
>500
>500
>1000
1000
>500
>500
>1000
>1000
>1000
500
0
>1000
>1000
>1000
>500
0
0
745 ± 5
187.5
1.5
0
4
500
727.5 ± 12.5
742.5 ± 27.5
>1000
>500
>500
>1000
>1000
>1000
500
375
500
2
500
>500
0
1.5
0
>1000
>1000
>1000
250
>1000
>1000
1000
250
>1000
>1000
>1000
>500
0
>1000
0
0
>1000
0
>1
512.5 ± 12.5
1
2
a
b
c
Maximum tolerable concentration (MTC) is the highest concentration of a compound that does not cause death of Vero E6 cells.
CD50 is the 50% cytotoxic concentration of a compound, required to reduce the viability of Vero E6 cells by 50%.
ID50 is the 50% effective concentration of a compound, achieving 50% protection of Vero E6 cells against the cytopathic effect of HSV-1.
ID95 is the 95% effective concentration of a compound, achieving 95% protection of Vero E6 cells against the cytopathic effect of HSV-1.
PFU is the plaque-forming unit.
d
e
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4
00
Synthesis
M. I. Kharitonova et al.
Paper
subtoxic concentrations of 250 and 500 μg/mL (MOI 0.01
and 0.1 PFU/cell respectively). For all those nucleosides, in-
dex selectivity (IS) values proved to be less than or equal to
creased cytotoxicity or to the appearance of antiviral activi-
ty. We did not find a clear correlation between the struc-
ture and activity among nucleosides synthesized. It is clear
that a more detailed study is required to elucidate behavior
of the active compounds relative to other viral models.
2
(see details in Table 5). The antiviral activity of 4-meth-
oxy-5,6-difluoro-1-(β-D-deoxyribofuranosyl)benzimidazole
24) was more selective (IS 1.5 and 4.0 for MOI 0.1 and 0.01
(
PFU/mL, respectively). The most selective antiherpes activi-
ty was detected for 4,5,6-trifluoro-1-(β-D-ribofurano-
syl)benzimidazole (21), for which the ID50 was found to be
All chemicals and solvents were of laboratory grade, as obtained from
commercial suppliers and were used without further purification.
The method used to produce recombinant E. coli phosphorylases was
2
50.0 and 62.5 μg/mL (MOI 0.1 and 0.01), IS 2.00 and 8.68,
respectively.
The enzymatic transglycosylation reaction appears to be
31
30
described previously. The following recombinant E. coli enzymes
were used in the present study: UP with specific activity 100 units
per mg of protein, 9 mg per mL; PNP 54 units per mg, 27 mg per mL;
the solutions of NPs were in 5 mM potassium phosphate buffer
(pH 7.0).
one of the most convenient methods for the synthesis of
benzimidazole nucleosides. By using this approach, we
have succeeded in synthesizing a series of ribo- and 2′-de-
oxyribosides of 5-substituted 4,6-difluorobenzimidazoles
The progress of the reactions and the purity of compounds were
monitored by TLC analysis (Sorbofil, Merck) in CHCl –EtOH, 10:1. For
3
1
1, 12 and 15–18. Similar to the synthesis of the previous
compounds 11a–d and 13–18, satisfactory elemental analysis was
obtained (C, H, N), ±0.40. Silica gel C18 (Merck) was used for reverse-
phase chromatography. HPLC was performed with a Waters system
series of benzimidazole nucleosides 6, N3-isomers of benz-
imidazole nucleosides (up to 18%) have been found in the
reaction mixtures, although the formation of N3-isomers
did not occur in the synthesis of nucleosides with a strong
electron-donating substituent (morpholino) in the 5-posi-
tion of the benzene ring.
The antiviral activity of nucleosides 19–30 against her-
pes simplex virus type 1 and their cytotoxicity has been
studied in cultures of Vero E6 cells. Despite incorporation of
an additional F-atom at the C4 position of 5,6-disubstituted
benzimidazoles, all studied compounds exhibited low cell
culture cytotoxicity (512.5–1000 μg/mL). Compound 24
demonstrated a reliable antiviral effect in concentrations
that were 1.5–4 times lower (MOI 0.1 and 0.01 PFU/cell, re-
(Waters 1525, Waters 2487, Breeze 2; USA); column: Nova Pack C18,
4
0
μm, 4.6 × 150 mm; eluent А: 0.1% TFA–H O, eluent В: 70% MeCN in
2
.1% TFA–H O; flow: 1 mL/min; UV detection: 254 and 280 nm. The
2
UV spectra were recorded with a Shimadzu UV-160 (Japan) spectro-
photometer. Mass spectra were recorded with an Agilent 6224, ESI-
1
TOF, LC/MS (USA) in positive ion mode (ESI+). H NMR spectra of com-
pounds 13–18 were recorded with a Bruker Avance II spectrometer
(
Bruker, Germany) (400 MHz) in DMSO-d . Chemical shifts in ppm (δ)
6
are reported relative to TMS as internal standard. NMR spectra of 19–
0 were recorded with a Bruker Avance-700-DRX spectrometer
Bruker, Germany).
3
(
Physicochemical properties and H and 13C NMR data are presented in
1
Table 6 and Table 7.
spectively) than the CD , whereas 4,5,6-trifluorobenzimid-
50
3,5-Difluoro-1,2-phenylenediamine (9)
azole riboside 21 exhibited the most selective anti-herpes
activity in this series: IS 2 (MOI 0.1 PFU/cell) and 8.7 (MOI
Concentrated hydrochloric acid (10 mL) was slowly added dropwise
to a mixture of difluoronitroaniline 7 (1.6 g, 9 mmol) and tin (22.5
mmol). The reaction mixture was heated at reflux for 1.5 h, cooled to
r.t., and neutralized with 50% NaOH solution to pH 9–10. The precipi-
tate was filtered, dried, and extracted with hot EtOAc (80 mL). After
evaporation of the filtrate, diamine 9 was obtained.
0.01 PFU/cell). At low MOI, both compounds fully inhibited
the progression of virus-induced cytopathic effect (CPE).
Thus, some clear relationships between antiherpetic ac-
tivity and the structure of BzIm-nucleosides can be ob-
served. Incorporation of a bulky substituent (morpholino)
in the C5-position results in complete supression of antivi-
ral activity (see the data for compounds 27 and 28). The an-
tiviral activity of compounds with a less bulky substituent,
such as the ethoxy (29 and 30) and pyrrolidino (23 and 24)
groups proved to enhance up to 1–2 IS. The behavior of
compounds 23 and 24 was unpredictable: the activity of ri-
boside dropped to 0, whereas that of deoxyriboside in-
creased up to 4 IS units. Activity of 4,6-difluoro-BzIm-nu-
cleosides was lower than that of 5-methoxy-4,6-difluoro
analogues: the IS of 20 and 19 was 0 and 1.5, respectively. In
contrast, the antiviral activity of 4,5,6-trifluoro-BzIm-de-
oxyriboside dropped to zero. Riboside 21 proved to be the
most active in the series of compounds (IS 8.68). From these
data we can make the following conclusions. Introduction
of an additional fluorine atom into the molecule changes
the properties of nucleosides but it does not lead to in-
Yield: 0.60 g (46%); brown crystals; mp 55–57 °C; R 0.86.
f
4,6-Difluorobenzimidazole (11)
Compound 9 (0.6 g, 4.2 mmol) was heated at reflux in formic acid (30
mL) for 4 h. The reaction mixture was evaporated under vacuum, and
the residue was recrystallized from H O–EtOH (1:2).
2
Yield: 0.5 g (78%); hazel crystals; mp ≥ 190 °C (Lit.22 ≥190 °C); R =
f
0.57.
3-Alkoxy-2,4-difluoro-6-nitroanilines (13a,b)
To solution of 2,3,4-trifluoro-6-nitroaniline (8; 1.54 g, 8 mmol) in al-
cohol (20 mL), a solution of KOH (0.45 g, 8 mmol) in the same alcohol
(20 mL) was added. The reaction mixture was heated at reflux for 1.5
h, cooled to r.t., and poured into ice water (120 vol). The precipitate
was filtered off, dried, and recrystallized (EtOH).
2,4-Difluoro-3-methoxy-6-nitroaniline (11a)
Yield: 1.30 g (80%); yellow crystals; mp 100–102 °C; R 0.83.
f
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4
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Synthesis
M. I. Kharitonova et al.
Paper
2
,4-Difluoro-3-ethoxy-6-nitroaniline (11b)
Enzymatic Reactions
Reaction conditions for the synthesis of benzimidazole nucleosides
ratio of reagents, the quantity of NPs, the molarity of the buffer, the
Yield: 1.23 g (70%); yellow crystals; mp 85–87 °C; R 0.80.
f
(
timing of the process, and amount of the starting substrate) are
shown in Table 4. The starting heterocyclic bases and nucleosides
were dissolved in potassium dihydrogen phosphate buffer solution
2
,4-Difluoro-3-(morpholino)- (11c) and 2,4-Difluoro-3-(pyrrolidi-
no)-6-nitroaniline (11d)
A mixture of 2,3,4-trifluoro-6-nitroaniline (8; 2.30 g, 12 mmol) and
morpholine (2.8 mL, 36 mmol) or pyrrolidine (3 mL, 36 mL) in EtOH
(pH 7.0) on heating to 40–50 °C. Enzymes were added and the reac-
tion mixture was incubated at 50 °C. HPLC was used to monitor the
progress of the reaction. The reactions that included 2′-deoxyribo-
sides were quenched by adding of EtOH (50% v/v) at the point of the
maximal conversion of base into nucleoside. The reaction mixtures
were concentrated to 5 mL. Nucleosides 21 and 22 were crystallized
from the reaction mixture after concentration to nearly five times.
Other products were isolated from mixtures by column chromatogra-
(
30 mL) was stirred at r.t. for 1 h. The precipitate was filtered off and
recrystallized (EtOH).
2
,4-Difluoro-3-(morpholino)-6-nitroaniline (11c)
Yield: 2.50 g (80%); yellow crystals; mp 158–160 °C; R 0.87.
f
2,4-Difluoro-3-(pyrrolidino)-6-nitroaniline (11d)
phy (C18, 20 × 100 mm, elution by gradient of H O–lipophilic solvent
2
such as MeCN, EtOH or MeOH, see Table 4). Physicochemical proper-
ties and spectral characteristics of the nucleosides are shown in Table
Yield: 2.45 g (88%); yellow crystals; mp 172–175 °C; R 0.85.
f
6
and Table 7.
3,5-Difluoro-4-methoxy- (14a) and 3,5-Difluoro-4-ethoxy-1,2-
phenylenediamine (14b)
Investigation of the Antiviral Activity and Cytotoxicity
A mixture of 13a–b (7 mmol), hydrazine hydrate (98%, Sigma–
Aldrich, 10 mL) and Raney Ni (80%, Sigma–Aldrich, 10% by weight of
the compound 13a–b) in EtOH (30 mL) was heated at reflux for 1 h. A
hot reaction mixture was filtered and the precipitate was washed
with EtOH (20 mL). The filtrate was evaporated and cooled to 4 °C,
and the dark-brown precipitate was filtered off, washed with EtOH,
and dried.
Continuous cell line cultures of green monkey kidney Vero E6 were
used to evaluate the activity of benzimidazole nucleosides 19–30
against herpes simplex virus type 1 (HSV-1) and their cytotoxicity.
Monolayer cell cultures were grown in 96-well plastic plates. The 24 h
monolayer cell culture was used. Culture conditions: 37 °C in 5% CO2
atmosphere.
HSV-1 strain L2 was obtained from the State Museum collection of vi-
ruses of D. I. Ivanovski Institute of Virology, Russian Ministry of
Health.
3
,5-Difluoro-4-methoxy-1,2-phenylenediamine (14a)
Yield: 1.00 g (84%); brown crystals; mp 65–67 °C; R 0.66.
f
Investigation of antiherpes activity of the compounds in vitro was
carried out in accordance with the internationally accepted cytopath-
3
,5-Difluoro-4-ethoxy-1,2-phenylenediamine (14b)
31–33
ic effect (CPE) inhibition assay.
Flow Laboratories’, UK) with the generated cell monolayer, serial di-
In 96-well plastic plates (Linbro,
Yield: 0.900 g (70%); brown crystals; mp 70–72 °C; R 0.66.
f
‘
lutions of the compounds were prepared with a multiplicity of 2 in
the support medium (Eagle medium and medium 199, 1:1). Infec-
tious material was then introduced with final dilution providing a
multiplicity of infection (MOI) 0.1 PFU/cell or 0.01 PFU/cell; incuba-
tion was performed at 37 °C. Each preparation was examined in two
parallels at each MOI. Three controls were used. 1) Toxicity control.
Uninfected cells were passaged in the presence of the drug in the
same concentration range as in the experiment. 2) Virus control. Cells
were infected under conditions described above, but the support me-
dium did not contain the drug. 3) Cells control. Uninfected cell cul-
tures were incubated in the support medium, which contained no
drug.
3
,4,5-Trifluoro- (10), 3,5-Difluoro-4-morpholino- (14c), and 3,5-
Difluoro-4-pyrrolidino-1,2-phenylenediamine (14d)
Concentrated HCl (15 mL) was slowly added dropwise to a mixture of
nitroaniline 8, 13c–d (12 mmol), and tin (30 mmol). The reaction
mixture was heated at reflux for 1.5 h, cooled to r.t., and neutralized
with 50% NaOH solution to pH 9–10. The precipitate was filtered off,
dried, and extracted with hot EtOAc (80 mL). Diamines 10 and 14c–d
were obtained after evaporation of filtrates.
3,4,5-Trifluoro-1,2-phenylenediamine (10)
Yield: 1.40 g (70%); brown crystals; mp 85–87 °C; R 0.72
f
.
After 48 and 72 h (for MOI 0.1 and 0.01 PFU/mL, respectively), when
95–100% CPE was developed in virus control, an antiviral activity was
assessed by determining of ID50 and ID95 for compound concentra-
tions that inhibit the development of virus-induced CPE as compared
with control by 50% and almost completely (Table 5).
3
,5-Difluoro-4-morpholino-1,2-phenylenediamine (14c)
Yield: 1.37 g (50%); brown crystals; mp 118–120 °C; R 0.85.
f
3,5-Difluoro-4-pyrrolidino-1,2-phenylenediamine (14d)
Cytotoxic properties of the compounds in vitro were evaluated after
Yield: 1.20 g (45%); dark-brown oil; R 0.85
f
72 h incubation of cells in the presence of a known concentration of a
compound. A maximal tolerable concentration (MTC) of compound
that does not cause destructive changes in the cell monolayer in com-
parison with controls was determined by the state of a cell monolay-
er. Cytotoxicity was determined quantitatively by the trypan blue ex-
clusion method and expressed as CD50: the concentration of the drug
4
,5,6-Trifluorobenzimidazole (12) and 5-Substituted 4,6-Difluoro-
benzimidazoles (15–18)
Compounds 10 and 14a–d (6 mmol) were heated at reflux in formic
acid (30 mL) for 4 h. The reaction mixture was then evaporated under
vacuum, poured into H O (50 mL) and neutralized with a 10% solution
of NaOH (20 mL) to pH 7. The precipitate was filtered off, dried, and
34–37
2
at which 50% of cells die.
recrystallized from a mixture H O–MeCN (1:1).
2
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Synthesis
M. I. Kharitonova et al.
Paper
Table 6 Physicochemical Properties of Nucleosides 19–30
Compound
Yield (%),
[mg]
Purity (%)
Mp (°C) (solvent),
physical appearance
UVb
max (nm)[ε]
Calcd [M + H]+ MSc
a
a
(HPLC)
[t
R
(min)]
λ
+
1
9
60
[77]
96.8
[7.1, 8.1 (II)]
–
–
–
–
287.0843
271.0894
305.0749
289.0800
317.0949
301.1000
331.1106
315.1156
372.1371
356.1422
356.1422
287.0858 [M + H] ,
(
19a/19b = 92:8)
white amorphous powder
155.0439 [M-Rib + 2 H]+
+
2
0
94
[116]
100.0
[6.7, 6.2 (II)]
–
271.0904 [M + H] ,
(
20a/20b = 94:6)
white amorphous powder
55.0420 [M-dRib + 2 H]+
+
2
1
89
[110]
96.5
[8.6, 8.0 (II)]
118–119 (H
colorless crystals
2
O)
305.0754 [M + H] ,
(
21a/21b = 90:10)
173.0340 [M-Rib + 2 H]+
+
2
2
65
[76]
98.9
[10.2, 11.2 (II)]
195–196 (H O)
colorless crystals
2
268 [3,900],
241 [7,400]
289.0794 [M + H] ,
(
22a/22b = 99:1)
173.0337 [M-dRib + 2 H]+
+
2
3
79
[95]
93.1
[9.0, 8.4 (II)]
–
–
317.0942 [M + H] ,
(
23a/23b = 82:18)
white amorphous powder
185.0540 [M-Rib + 2 H]+
+
2
4
83
[95]
99.1
[9.4 (II)]
–
274 [3,800],
243 [6,600]
301.0966 [M + H] ,
(
24a/24b = 90:1)
white amorphous powder
185.0517 [M-dRib + 2 H]+
+
2
5
70
[82]
97.9
[17.0, 18.0 (I)]
–
–
331.1097 [M + H] ,
(
25a/25b = 93:7)
white amorphous powder
199.0690 [M-Rib + 2 H]+
+
2
6
71
[79]
86.2
[11.4, 11.2 (II)]
–
–
315.1120 [M + H] ,
(
26a/26b = 94:6)
white amorphous powder
199.0665 [M-dRib + 2 H]+
2
2
2
7a
8a
9
83
97.6
[9.5 (II)]
–
277 [3,500]
277 [3,700]
372.1342 [M + H]+,
[
88]
white amorphous powder
240.0930 [M-Rib + 2 H]+
93
98.3
[9.1 (II)]
–
356.1395 [M + H]+,
[
89]
white amorphous powder
240.0930 [M-dRib + 2 H]+
+
61
[68]
84.7
[6.2, 6.4 (II)]
–
305 [1,400],
231 [16,400]
356.1422 [M + H] ,
(
29a/29b = 98:2)
white amorphous powder
224.0981 [M-Rib + 2 H]+
(
EtOH)
+
3
0
63
[56]
92.5
[8.5, 10.7 (II)]
–
305 [2,000];
231 [18,500]
340.1473
340.1467 [M + H] ,
(
30a/30b = 95:5)
white amorphous powder
224.0999 [M-dRib + 2 H]+
(
EtOH)
a
Based on HPLC analysis performed with a Waters system (Waters 1525, Waters 2487, Breeze 2) using column Nova Pack C18, 4 μm, 4.6 × 150 mm. Eluent А:
0
.1% ТFA/H O, eluent В: 70% MeCN in 0.1% ТFA/H O. Flow: 1 mL/min, UV detection at 254 and 280 nm. Elution: (I) 0 to 30% В over 20 min, (II) 0 to 70% В over 20
2
2
min.
b
The UV spectra were recorded with a Shimadzu UV-160 (Japan) spectrophotometer in H
2
O, if not indicated otherwise.
c
Mass spectra were recorded with an Agilent 6224, ESI-TOF, LC/MS (USA).
Table 7 Selected H and C NMR Spectroscopic Data of Benzimidazole Nucleosides (DMSO-d , δ, ppm, J, Hz)a
1
13
6
Compd H-1′
C1′]
H-2′a
[C2′]
H-2′b
[C2′]
H-3′
[C3′]
H-4′
[C4′]
H-5′a/H-5′b Others
[C5′]
[
[
13C]{15N}
3
1
9a
5.86 (d, J = 4.33 (m, J = 6.0,
–
4.12 (m)
[70.37]
3.99 (m)
[86.22]
3.63, 3.68
(m)
[61.47]
8.50 (s, 1 H, H-2) [144.06], 7.63 (dd, J
=
H7,F6
4
6.4)
5.6)
8.98, J
= 1.2, 1 H, H-7) [95.77], 7.11 (dt,
JH5,F4 = 3JH5,F6 = 10.5, 4JH5,H7 = 1.3, 1 H, H-5)
H7,H5
3
[
89.46]
[74.02]
[
98.07], 5.45 (d, J = 6.4, 1 H, 2′-OH), 5.20 (d,
J= 4.5, 1 H, 3′-OH), 5.17 (t, J = 5.0, 1 H, 5′-
OH), [158.51 (d, JCF = 235, C6), 152.98 (d,
JCF = 270 C4), 135.4 (s, C8), 129.47 (s, C9)],
{
237.36 (N3), 167.90 (N1)}
3
1
9b
5.98 (d, J = 4.36 (m)
5.5) [75.15]
89.98]
–
4.09 (m)
[70.20]
3.95 (m)
[85.68]
3.57, 3.68
(m)
[61.25]
8.68 (s, 1 H, H-2) [144.73], 7.40 (dd, J
=
H4,F5
4
7%
9.4, J
(dd, J
= 1.35, 1 H, H-4) [102.24], 7.22
H4,H6
3
3
4
[
= J
=10.9, J
< 1.4, 1 H, H-
H6,F4
H5,F7
H4,H6
6) [99.53], 5.50 (d, J = 6.5, 1 H, 2′-OH), 5.04
(
t, J = 5.0, 1 H, 5′-OH)
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4
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Synthesis
M. I. Kharitonova et al.
Paper
Table 7 (continued)
Compd H-1′
H-2′a
[C2′]
H-2′b
[C2′]
H-3′
[C3′]
H-4′
[C4′]
H-5′a/H-5′b Others
[C5′]
[
C1′]
[
13C]{15N}
2.55 (m, gemJ =
2.30 (m, gemJ = 4.40 (m)
13.3, 6.1, 3.3) [70.69]
[40.1]
3.88 (m)
[88.09]
3.56, 3.60
(m)
[61.70]
8.52 (s, 1 H, H-2) [143.7], 7.58 (dd, J
3
=
2
0a
6.34 (dd,
J = 6.3, 7.1) 13.3, 7.1, 1.1)
H7,F6
4
9.0, J
= 2.0, 1 H, H-7) [95.48], 7.10 (dt,
JH5,F4 = 3JH5,F6 = 10.7, 4JH5,H7 = 2.1, 1 H, H-5)
H7,H5
3
[
85.40]
[40.1]
[
98.0], 5.32 (d, J = 3.7, 1 H, 3′-OH), 5.0 (t, J =
5.0, 1 H, 5′-OH), [135.55 (s, C8), 129.38 (s,
C9)], {172.3 (N1), 237.5 (N3)}
2
0b
6.41 (dd,
2.58 (m)
[40.25]
2.37 (m)
[40.25]
4.34 (m)
[nd]
[nd]
3.50, 3.49
(m)
[nd]
8.62 (s, 1 H, H-2) [144.4], 7.38 (m, 1 H, H-4)
[nd], 7.20 (m, 1 H, H-5) [nd], 4.91 (br t, 1 H,
5′-OH)
5%
J = 5.5)
b
[
nd]
2
1a
5.87 (d, J = 4.31 (m)
.43) [74.03]
89.55]
–
4.12 (br m)
[70.47]
4.00 (br m)
[86.39]
3.65, 3.68
(m)
[61.45]
8.58 (s, 1 H, H-2) [145.14], 7.94 (dd, 3JH7,F
=
9.5, 6.4, 1 H, H-7,) [96.46], 5.46 (d, J = 6.4,
1 H, 2′-OH), 5.22 (d, J = 4.2, 1 H, 3′-OH),
6
[
5
.22 (t, J = 4.26, 1 H, 5′-OH), [147.1 (d,
JCF=230, C6), 129.3 (C8)], {236.67 (N3),
67.44 (N1)}
1
2
1b
5.97 (d, J = 4.36 (m)
–
4.10 (br s)
[nd]
3.96 (m)
[85.91]
3.57, 3.58
(m)
[nd]
8.70 (s, 1 H, H-2), 7.69 (m, 1 H, H-4), 5.46
(d, J = 6.0, 1 H, 2′-OH), 5.05 (t, J = 4.8, 1 H,
5′-OH)
4%
5.0)
90.07]
[75.08]
[
2
2а
6.35 (dd,
J = 6.9, 6.3) 13, 6.6)
2.54 (m, gemJ =
2.30 (m)
[40.20]
4.40 (m)
[70.68]
3.89 (m)
[88.20]
3.60–3.57 8.59 (s, 1 H, H-2) [144.76], 7.89 (dd, J =
(m)
10.0, 6.3, 1 H, H-7) [96.18], 5.33 (d, J = 4.0,
1 H, 3′-OH), 5.04 (t, J = 5.1, 1 H, 5′-OH),
[
85.57]
[40.20]
[61.62]
[
147.8 (d, C6, JCF=230), 140.1 (d, C5,
JCF=240), 129.25 (s, C8)], {237.0 (N3),
71.44 (N1)}
1
2
2b
6.41 (dd,
Overlap with
2.28 (m)
[nd]
4.35 (m)
[nd]
3.98 (m)
[nd]
3.49, 3.34
(m)
[nd]
8.64 (s, 1 H, H-2), 7.66 (dd, J = 10.4, 7.0, 1 H,
H-4), 5.34 (br d, 1 H, 3′-OH), 4.90 (t, J = 5.3,
1 H, 5′-OH)
<1%
J = 6.7, 6.4) signal H-2′a
nd] 22a
nd]
[
[
2
3a
5.83 (d, J = 4.31 (m, J =
–
4.11 (m)
[70.52]
3.99 (m)
[86.30]
3.64–3.67 8.48 (s, 1 H, H-2) [144.34, C2], 7.72 (d, 3JH7,F6
6.5)
10.7, 5.2)
[74.03]
(m)
= 10.8, 1 H, H-7) [96.03, C7, JC7,F6 = 26.8],
5.43 (d, J = 5.4, 1 H, 2′-OH), 5.20 (d, J= 4.4,
[
89.45]
[61.48]
1
3
H, 3′-OH), 5.18 (t, J = 5.0, 1 H, 5′-OH),
.90 (s, 3 H, OCH ) [62.91, CMe], [129.3 (d,
3
JC7a,F6 = 15.8, C7a), 129.65 (dd, J= 98.1, 14.5,
C5), 132.12 (dd, J = 17.3, 12.7, C4a), 146.58
(
2
dd, JC6,F6 = 253.5, 7.7, C6), 153.05 (d, JC6,F6
41.5, C6)], {236.8 (N3), 166.5 (N1)}
=
2
2
3b
4a
5.96 (d, J = 4.36 (m)
.5) [74.15]
89.84]
–
4.09 (m)
[70.26]
3.95 (m)
[85.74]
3.63, 3.57
(2 m)
[61.44]
8.61 (s, 1 H, H-2) [144.09], 7.48 (d, 3JH4,F5
=
10.58, 1 H, H-4) [102.64], 5.51 (br d, 1 H, 2′-
OH), 1 H, 3′-OH overlap with 3′-OH signal of
5
[
2
3a, 5.04 (t, J = 4.5, 1 H, 5′-OH), 3.93 (s,
3
H, OCH ) [62.97]
3
6.32 (dd,
2.54 (m, J = 12, 2.28 (m, J =
4.39 (m)
[80.70]
3.87 (m)
[88.08]
3.56–3.58 8.49 (s, 1 H, H-2) [143.79], 7.67 (d, 3JH7,F6
=
(m)
[61.67]
J = 6.3, 7.4) 7.4)
12, 6.34)
[40.04]
10.4, 1 H, H-7,) [95.64], 5.31 (d, J= 4.1, 1 H,
3′-OH), 5.00 (t, J = 5.1, 1 H, 5′-OH), 3.90 (s,
[
89.45]
[40.04]
3
H, OCH ) [62.77], [129.5 (s, C8), 132.1 (s,
3
2
C5), 152.9 (d, J
= 243, C6)], {236.85
C6,F6
(
N3), 170.82 (N1)}
2
<
4b
1%
6.40 (br t,
J ~ 6.5)
ca. 2.54 (m),
[nd]
ca. 2.28 (m)
[nd]
4.34 (m)
[nd]
ca. 3.87 (m)
[nd]
ca. 3.56–
3.58 (m)
[nd]
8.65 (s, 1 H, H-2), 7.46 (d, 3JH4,F6 = 10.5, 1 H,
H-4), 5.37 (br sign, 1 H, 3′-OH), 4.91 (t, J =
5.5, 1 H, 5′-OH), 3.93 (s, 3 H, OCH3)
[
nd]
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 394–406

4
04
Synthesis
M. I. Kharitonova et al.
Paper
Table 7 (continued)
Compd H-1′
H-2′a
[C2′]
H-2′b
[C2′]
H-3′
[C3′]
H-4′
[C4′]
H-5′a/H-5′b Others
[C5′]
13C]{15N}
[
C1′]
[
2
5а
5.83 (d, J = 4.31 (m)
.4) [73.91]
89.45]
–
4.11 (m)
[71.10]
3.99 (m)
[86.24]
3.64–3.67 8.47 (s, 1 H, H-2) [144.20], 7.71 (d, 3JH7,F6
=
6
[
(m)
10.5, 1 H, H-7,) [95.80], 5.43 (d, J = 6.4, 1 H,
[61.49]
2′-OH), 5.19 (d, J= 4.8, 1 H, 3′-OH), 5.18 (t,
J = 4.6, 1 H, 5′-OH), 4.12 (m, 2 H, OCH CH )
2
3
[
[
1
70.41], 1.31 (t, 3 H, CH CH ) [15.61],
2 3
129.3 (s, C8), 129.9 (s, C9), 130.66 (s, C5),
2
53.27 (d, J
= 236.87, C6)], {236.81
C6,F6
(
N3), 166.54 (N1)}
2
5b
5.96 (d, J = 4.36 (m)
–
nd over-
lapped with
3.94 (m)
[nd]
ca. 3.57
(m)
[nd]
8.61 (s, 1 H, H-2), 7.47 (d, 3JH4,F6 = 10.4, 1 H,
H-4), 5.50 (d, J = 5.9, 1 H, 2′-OH), ca. 5.19
(overlapped, 1 H, 3′-OH), 5.04 (t, J = 5.1,
1 H, 5′-OH), 4.15 (m, 2 H, OCH CH ), 1.32
6%
5.2)
89.75]
[nd]
[
the sign. 3′-H
5a
2
2
3
(
t, 3 H, CH CH )
2 3
2
6а
6.31 (dd,
J = 6.4, 7.3) 13.5, 6.8)
2.54 (dt, J =
2.28 (ddd, J = 4.39 (m)
13.3, 5.8, 3.2) [70.80]
[40.51]
3.87 (m)
[88.20]
3.56–3.59 8.49 (s, 1 H, H-2) [143.88], 7.66 (d, 3JH7,F6
=
(m)
10.3, 1 H, H-7) [95.63, d, JC7,F6 = 26.0], 5.31
(d, J = 4.0, 1 H, 3′-OH), 5.0 (t, J = 5.10, 1 H,
′-OH), 4.12 (dt, J = 7.10, 2 H, CH CH )
[
85.48]
[40.51]
[61.77]
5
2
3
[
[
70.99], 1.31 (t, J = 7.0, 3 H, CH CH )
2 3
15.64], [129.5 (dd, JC7a,F6 = 9.4, 14.7, C7a),
1
1
1
1
29.66 (d, JC4a,F5 = 14.3, C4a), 130.66 (dd, J =
1.0, 16.9, C5), 146.90 (d, J
= 254.6, C4),
C4,F4
2
53.17 (d, J
= 240.7, C6)], {236.65 (N3),
C6,F6
70.89 (N1)}
2
6b
6.40 (br t,
J = 6.48)
2.57 (m)
[nd]
2.36 (m)
[nd]
4.34 (m)
[nd]
3.87 over-
lapped with
3.46–3.56 8.56 (s, 1 H, H-2) [143.80], 7.45 (d, 3JH4,F6
(m) 10.8, 1 H, H-4) [102. 25], ca. 5.31 (over-
=
6%
[
nd]
the sign. 4′-H [nd]
6a
nd]
lapped, 1 H, 3′-OH), 5.0 (t, J = 5.48, 1 H, 5′-
OH), 4.12 (dt, J = 7.20, 2 H, CH CH ), 1.32 (t,
overlapped with CH 26a, 3 H, CH CH )
3 2 3
2
[
2
3
2
7a
5.82 (d, J = 4.30 (m)
–
4.10 (m)
[70.42]
3.98 (m)
[86.19]
3.61–3.70 8.44 (s, 1 H, H-2) [143.95], 7.61 (d, 3JH7,F6
=
6.0)
[73.98]
(m)
10.9, 1 H, H-7,) [95.83], 5.43 (d, J = 6.0, 1 H,
[
89.45]
[61.50]
2′-OH), 5.19 (d, J = 3.5, 1 H, 3′-OH), 5.16 (t,
J = 4.5, 1 H, 5′-OH), 3.71 (br s, 4 H, O(CH ) )
2
2
[67.35], 3.10 (br s, 4 H, N(CH ) ) [52.0],
2 2
[
122.84 (C5), 130.02 (C4a), 130.82 (C7a),
50.03 (d, JC4,F4 = 244.2, C4), 156.47 (d,
JC6,F6 = 243.2, C6)], {166.41 (N1), 237.54
N3), 287.19 N-morph}
1
(
2
8a
6.30 (t, J =
.7)
85.28]
2.53 (m, J =
13.6, 6.8)
[40.24]
2.28 (m, J =
13.2, 5.8, 3.2) [70.66]
[40.24]
4.39 (m)
3.87 m
[88.08]
3.55–3.58 8.46 (s, 1 H, H-2) [143.55], 7.56 (d, 3JH7,F6
=
6
[
(m)
11.2, 1 H, H-7) [95.65], 5.31 (d, J = 3.9, 1 H,
3′-OH), 4.99 (d, J = 5.2, 1 H, 5′-OH), 3.71
[61.61]
(
m, 4 H, CH OCH ) [67.24], 3.09 (m, 4 H,
2 2
CH NCH ) [51.82], [122.76 (C5), 129.8
2
2
(
(
(
C4a), 150.10 (d, JC4,F4 = 237.8, C4), 156.52
d, JC6,F6 = 248.2, C6)], {170.91 (N1), 237.69
N3), 287.23 (N-morph)}
2
9a
5.79 (d, J = 4.30 (m)
.3) [73.77]
89.34]
–
4.10 (m)
[70.39]
3.97 (m)
[86.02]
3.63–3.67 8.39 (s, 1 H, H-2) [143.45], 7.55 (d, 3JH7,F6
=
6
[
(m)
11.9, 1 H, H-7) [95.66], 5.41 (d, J = 6.5, 1 H,
[61.50]
2′-OH), 5.18 (d, J= 4.5, 1 H, 3′-OH), 5.15 (t,
J = 4.9, 1 H, 5′-OH), 3.30 (m, 4 H, CH NCH )
2
2
[51.82], 1.90 (m, 4 H, NCH (CH ) ) [25.61],
2 2 2
[
121.32 (s, C5), 128.93 (s, C8), 130.34 (s,
2
C9), 148.42 (d, J
2
= 247, C4), 155.52 (d,
C4,F4
JC6,F6 = 242, C6)], {236.70 (N3), 165.65 (N1),
62.66 (NPyr)}
1
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 394–406

4
05
Synthesis
M. I. Kharitonova et al.
Paper
Table 7 (continued)
Compd H-1′
H-2′a
[C2′]
H-2′b
[C2′]
H-3′
[C3′]
H-4′
[C4′]
H-5′a/H-5′b Others
[C5′]
[
C1′]
[
13C]{15N}
2
9b
5.95 (d, J = 4.35 (m)
–
4.20 (m)
[nd]
3.92 (m)
[nd]
ca. 3.56
(m)
[nd]
8.50 (s, 1 H, H-2), 7.32 (d, 3JH4,F5 = 12.18,
1 H, H-4), 5.46 (d, J = 5.8, 1 H, 3′-OH), 5.02
(t, J = 5.2, 1 H, 5′-OH), H-pyr overlapped
with signals of 29a 3.30 (m, 4 H, CH NCH )
<1%
5.4)
nd]
[nd]
[
2
2
and 1.90 (m, 4 H, NCH (CH ) )
2
2 2
3
0a
6.28 (dd,
J = 6.8)
2.53 (m, J =
13.2, 5.86, 3.2) 13.4, 6.98,
[40.02]
2.26 (m, J =
4.38 (m)
[70.73]
3.85 (m)
[88.0]
3.55–3.57 8.40 (s, 1 H, H-2) [143.28], 7.49 (d, 3JH7,F6
=
(m)
11.7, 1 H, H-7) [95.57], 5.30 (d, J = 2.5, 1 H,
3′-OH), 4.98 (t, J = 4.8, 1 H, 5′-OH), 3.30 (m,
4 H, CH NCH ) [51.88], 1.89 (m, 4 H,
[
85.28]
3.27)
40.02]
[61.71]
[
2
2
NCH (CH ) ) [25.67], [121.32 (dd, J = 9.6,
2
2 2
16.4, C5), 129.09 (dd, J = 10.06, 16.6, C7a),
130.25 (d, J = 17.0, C4a), 148.50 (dd, J =
253.0, 9.5, C4), 155.7 (dd, J = 241.4, 6.9,
C6)] {170.02 (N1), 236.98 (N3), 292.61
NPyr)}
(
3
0b
6.39 (br t,
J ~ 6.22)
ca. 2.54 (m)
[nd] over-
lapped with
signals of 30a
ca. 2.27 (m)
[nd] over-
lapped with
signals of 30a
4.34 (m)
overlapped
with sign [nd]
3.91 (m)
[nd]
3.51 (m)
[nd]
8.46 (s, 1 H, H-2), 7.31 (d, 3JH4,F5 = 12.5, 1 H,
H-4), 5.38 (br s, 1 H, 3′-OH), 4.91 (br t, J ~
4.8, 1 H, 5′-OH), 3.33 (m, 4 H, CH NCH )
3%
[
nd]
2
2
overlapped with H O signal, ca. 1.89 (m, 4 H,
2
NCH (CH ) ) overlapped with signals of 30a.
2
2 2
a
The NMR spectra were recorded with a Bruker Avance-700-DRX (Bruker, Germany). H, 13C, and 15N resonances were assigned using two-dimension-
1
1
1
1
13
1
13
1
15
1
15
1
1
al homo- and heteronuclear experiments: H/ H-COSY, H/ C-HSQC, H/ C-HMBC, H/ N-HSQC, H/ N-HMBC and [ H, H] NOE spectra.
b
nd] – not determined; for example, the quantity of isomer 20b (5%) in a mixture with 20a was not sufficient to determine the shifts of its 13C
[
1
13
1
13
nucleus in H/ C-HSQC, H/ C-HMBC spectra.
(
8) Krosky, P. M.; Borysko, K. Z. Antimicrob. Agents Chemother. 2002,
Acknowledgment
2
, 478.
The authors are thankful to the Ministry of Industry and Trade of the
Russian Federation (Minpromtorg of Russia) (State Contract No.
(9) Averyl, R. K.; Marty, F. M.; Strasfeld, L.; Lee, I.; Arrieta, A.; Chou,
S.; Tatarowicz, W.; Villano, S. Transplant Infect. Dis. 2010, 12,
489.
11411.1003702.13.050) and the Ministry of Education of the Russian
Federation (Project No. 2458) for the financial support of this work.
We are indebted to Dr. Tamara A. Balashova (Institute of Bioorganic
Chemistry, Moscow) for assistance in the analysis of NMR spectra of
new nucleosides. Dr. Roman S. Esipov for recombinant nucleoside
phosphorylases for experiments.
(10) Trofe, J.; Pote, L.; Wade, E.; Blumberg, E.; Bloom, R. D. Ann. Phar-
macother. 2008, 42, 1447.
(11) Alain, S.; Revest, M.; Veyer, D.; Essig, M.; Rerollers, J. P. Trans-
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