Design, synthesis, and biological evaluation of novel coxsackievirus B3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.04.081

The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66-2 μM). Minimal or no cytotoxicity was observed.

Full text of DOI:10.1016/j.bmc.2010.04.081

Bioorganic & Medicinal Chemistry 18 (2010) 4374–4384
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel coxsackievirus
B3 inhibitors
a
b
a,
a
a
*
ˇ
´
ˇ
´
Michal Šála , Armando M. De Palma , Hubert Hrebabecky , Radim Nencka , Martin Dracínsky ,
b
b
a
´
Pieter Leyssen , Johan Neyts , Antonín Holy
a Gilead Sciences & IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i, 166 10 Prague 6, Czech Republic
b Rega Institute for Medical Research, Minderbroedersstraat 10, BE-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These
compounds could be considered as the 6-chloropurines substituted at position 9 with variously substi-
tuted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene—norbornane or norbornene). The synthesis and bio-
logical evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in
Received 18 February 2010
Revised 21 April 2010
Accepted 25 April 2010
Available online 29 April 2010
the low micromolar range (0.66–2
l
M). Minimal or no cytotoxicity was observed.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
6-Chloropurine
Coxsackievirus B3
Mitsunobu reaction
Bicyclo[2.2.1]heptane
Fluorination
1. Introduction
broad-spectrum anti-enterovirus activity including activity against
Echo 9 at micromolar level unlike the parental compound MDL-
860. Anti-enterovirus activity in micromolar range was also re-
ported for compounds derived from 2,6-dihalophenyl-substituted
1H,3H-thiazolo[3,4a]benzimidazoles.8
Coxsackieviruses are non-enveloped, single-stranded (+) RNA
viruses belonging to the Picornaviridae family.1 Coxsackieviruses
are subdivided into two subgroups—CVA and CVB, based on their
pathology to newborn mice. Infections with coxsackieviruses,
and in particular with CVB3, are the most common cause of viral
myocarditis. Moreover, these viruses are reported to be associated
with the development of pancreatitis, meningitis, and encephali-
tis.2 Other diseases associated with coxsackievirus infections are
hand-foot-and-mouth disease and hemorrhagic conjunctivitis.
There is currently no approved antiviral therapy for the treat-
ment of picornaviral infections in man or animals. Numerous com-
pounds were reported in the past as selective inhibitors of
enteroviruses.3 For example, pleconaril entered clinical trials but
despite the promising results, it was not approved by the FDA for
the treatment of common cold symptoms in HRV-infected pa-
tients.4 Rupintrivir, another compound tested in the clinical trials,5
failed in natural infection study in patients.6 A large series of prom-
ising novel compounds based on substituted 5-nitro-5-pheno-
xybenzonitriles was recently discovered. Most of the compounds
examined in the study exerted significant activity against CVB3
Recently, we reported
a series of carbocyclic nucleoside
analogues with antiviral activity against coxsackieviruses. These
compounds carry bicycloalkanes,9 bicycloheteroalkanes10 or tricy-
cloheteroalkanes11 instead of the glycone part of the nucleoside
molecule (compounds 1–6, Fig. 1). The compounds were primarily
designed as the conformationally locked analogues of carbocyclic
nucleosides.9 Some of the analogues also exhibited antiviral activ-
ity against HIV-1 and HIV-2 on T-lymphocyte (CEM) cells.9b,c,11a
Activity against CVB3 was in the micromolar range with minimal
or no cytotoxicity in Vero cells (Table 1).
This study involves the synthesis of a second generation of this
compound class with bicyclic scaffold (variously substituted bicy-
clo[2.2.1]heptane) connected with a nucleobase (6-chloropurine)
at position 2. Our concept was based on the simplification of the
previously reported compounds with activity against CVB3.9–11
We examined which substitutions on the bicyclic part of the mol-
ecule are necessary for antiviral activity.
comparable with the parental analogue MDL-860 (6.5 4.9 l
M),7
and some of them were also tested against other picornaviruses.
The compound 2-(2-chlorophenoxy)-5-nitrobenzonitrile possessed
2. Synthetic chemistry
The first group of target compounds were synthesized by the
Mitsunobu reaction (Method A). 6-Chloropurine was coupled with
* Corresponding author. Tel.: +420 220 183 323; fax: +420 220 183 560.
ˇ
´
E-mail address: hubert@uochb.cas.cz (H. Hrebabecky).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.081

M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
4375
N
N
N
Cl
Cl
Cl
O
Cl
HO
HO
Cl
N
N
N
N
N
N
N
N
N
N
O
O
O
1
2
3
N
N
N
Cl
Cl
Cl
O
HO
N
N
N
N
HO
N
N
N
N
O
4
5
6
Figure 1. Structure of compounds 1–6.
Table 1
Table 2
B = 6-chloropurin-9-yl
Compound
IC50
(
l
M)
CC50 (lM)
Cl
N
1
2
3
4
5
6
1.13 0.33
1.86 1.13
1.00 0.04
0.88 0.11
0.98 0.17
0.91 0.07
>50
>50
>50
>50
>50
>50
N
N
N
R
OH
R
a
b
Compound
Alcohol (a)
Product (b)
Yield (%)
the appropriate alcohol (Table 2). The source of these alcohols is re-
ported in the Supplementary data. Only few derivatives were pre-
pared by construction of the nucleobase at the aminogroup on the
corresponding scaffold by a coupling with 4,6-dichloropyrimidin-
5-amine and the subsequent ring closure reaction12 (Method B, Ta-
ble 3, source of the amines—see Supplementary data). Yields are gi-
ven in Tables 2 and 3.
Several compounds with a hydroxy group at the scaffold were
also prepared and tested for the biological activity. Compound 22
with cis-dihydroxy arrangement was prepared from the unsatu-
rated derivative by the simple catalytic cis-hydroxylation with os-
mium tetroxide in high yield (81%). The oxirane ring was
introduced by epoxidation of the double bond with m-chloroper-
oxybenzoic acid, compound 23 was obtained in 73% yield (Scheme
1).
Derivatives bearing one hydroxy group were prepared from the
commercially available unsaturated acetylderivative 24, which
was hydroborated and the obtained mixture of the acetates were
immediately benzoylated (benzoyl chloride/pyridine) (Scheme 2).
The acetyl group was then selectively cleaved with potassium car-
bonate in methanol and the obtained alcohol was oxidized to a ke-
tone function with pyridinium dichromate (PDC) in
dichloromethane, yielding a mixture of ketones 25 and 26. At this
stage of the reaction sequence, the isomers were separated by col-
umn chromatography (overall yield from the starting acetate 24
was 30% for 25 and 16% for 26). Ketones 25 and 26 were reduced
with sodium borohydride in methanol to obtain the endo-hydroxy
compounds 27 and 28.
Benzoylated alcohols afforded protected chloropurine deriva-
tives 29 and 30 under Mitsunobu conditions (Scheme 3). The ben-
zoyl group was removed by a modified version of the Jeong et al.
method.13 Methyl magnesium chloride was used (4.4 equiv) and
the reaction time was prolonged. Cleavage of the benzoyl group
using classical methods (methanolic ammonia/4 °C, NaCN/metha-
nol, KCN/methanol, diluted aqueous sodium hydroxide/0 °C) was
not successful (Isolation of products was not achieved due to the
reaction giving a complicated mixture of products). In contrast,
only the previously published method9d with diisobutylaluminum
B
7
41
62
14
74
OH
OH
OH
B
B
8
9
OH
B
B
10
OH
11
12
13
68
35
33
B
OH
B
OH
B
14
15
16
17
18
46
78
65
65
75
OH
B
OH
B
OH
B
Ph
Ph
Ph
Ph
OH
B
OH
hydride in dichloromethane gave comparable yield, but isolation of
the products from the reaction mixtures appeared rather
complicated.

4376
M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
Table 3
B = 6-chloropurin-9-yl
BzO
O
O
BzO
BzO
i
Cl
N
25
ii
26
ii
OAc
N
N
R
NH2
N
24
R
BzO
a
b
OH
OH
Compound
Alcohol (a)
NH2
Product (b)
Yield (%)
71
28
27
B
Scheme 2. Reagents and conditions: (i) (1) BH3–THF, THF, 0 °C, 3 h, (2) NaBO3,
H2O–THF, rt, overnight, (3) BzCl, pyridin, rt, overnight, (4) K2CO3, MeOH, rt, 45 min,
(5) PDC, CH2Cl2, rt, overnight, 25 (30%), 26 (16%); (ii) NaBH4, MeOH, 0 °C, 1 h, 90%
for 27, 89% for 28.
19
O
O
B
NH2
20
21
65
40
OH
O
OH
N
Cl
BzO
BzO
N
RO
ii
i
N
NH2
B
N
OH
27
29
, R = Bz
31, R = H
Configuration of the hydroxy group was inverted to endo by
simple oxidation to the ketones 33/34 and subsequent reduction
of the keto group with sodium borohydride (Scheme 4). Inversion
of the configuration was not complete. A small portion of the
original exo-derivative 31 (17%) or 32 (20%) was observed in the
reaction mixture after reduction. Ketoderivatives 33 and 34 and
endo-hydroxy derivatives 37 and 38 were used to synthesize the
compounds 39 and 40 bearing the fluorine atom on the bicyclic
scaffold. Both fluorinations were carried out with (diethyl-
amino)sulfur trifluoride (DAST) in refluxing dichloromethane in
the presence of pyridine as a base. Geminal difluoroderivatives
35/36 and monofluoroderivatives 39/40 were prepared in moder-
ate to good yields (55% for 35; 45% for 36; 73% for 39; 63% for 40).
Another group of compounds that were synthesized are deriva-
tives bearing two chloropurine nucleobases. Compounds 41 and 42
with the bicyclic scaffold substituted at positions 2,6 or 2,5 and
exo-configuration of the substituents were prepared by the Mits-
unobu reaction. Endo-alcohols 37 and 38 were treated with chloro-
purine–triphenylphosphine–azodicarboxylate and moderate yields
of the final compounds were obtained (50% for 41, 55% for 42)
(Scheme 5).
Compounds bearing two bases in trans-configuration at posi-
tions 2 and 3 were prepared by construction of the nucleobase
on the appropriate amines (Scheme 6). The unsaturated diamine
4314 was coupled with 4 equivalents of the 4,6-dichloropyrimi-
din-5-amine and the reaction time was prolonged as compared
to the classical protocol used for the compounds 19b–21b. Despite
this, the yield of this construction was relatively low (19%). Similar
observations were obtained with the saturated diamine 44.15 In
this case, yield was 20%.
N
Cl
N
i
N
RO
N
OH
30
, R = Bz
28
ii
32, R = H
Scheme 3. Reagents and conditions: (i) 6-chloropurine, PPh3, DIAD, THF, rt to
reflux, 63% for 29, 48% for 30; (ii) CH3MgCl, THF, 0 °C, overnight, 58% for 31, 72% for
32.
13C signals is based on combination of 1H, 13C APT, H,H-COSY, H,C-
HSQC, and H,C-HMBC experiments. The position of the hydroxy or
keto group in compounds 25 and 26 was confirmed using HMBC
spectra. The hydrogen atom in position 2 (which is easily recog-
nized by its typical chemical shift 4.5–5.5 ppm) exhibits a three-
bond correlation cross-peak with carbon 6 and there is usually
no cross-peak with carbon 5. In the HMBC spectrum of 25, we ob-
served a strong cross-peak with a carbonyl carbon (at d 214.5),
which indicates the 2,6-substitution of the bicyclic system. On
the other hand, in the HMBC spectrum of 26, we did not observe
the cross-peak to the carbonyl carbon. Similar consecution was
used in case of the compounds 17 and 18. Configuration on carbons
2, 3, 5, and 6 was confirmed using 1H NMR coupling constants—the
endo-hydrogens do not interact with bridgehead hydrogens (dihe-
dral angle is close to 90°), while the exo-hydrogens interact with
coupling constant J = 4–5 Hz. Another significant interaction is be-
tween the endo-hydrogens in positions 2, 3, 5, and 6 and hydrogens
in position 7 (W-interaction, J = 1–2 Hz).
All target compounds were evaluated for antiviral activity
against coxsackievirus B3 (Table 4). TTP-8307 was included as a
reference compound.16 Antiviral activity was expressed by means
of EC50 or EC90 values, whereas CC50 values were calculated for
cytotoxicity. Most compounds efficiently inhibited coxsackievirus
B3 replication with minimal cytotoxicity to Vero cells. Only
3. Results and discussion
The structures of the prepared compounds were determined by
1H and 13C NMR spectroscopies. Complete assignment of all 1H and
N
N
N
Cl
Cl
Cl
HO
N
N
N
i
ii
O
N
N
HO
N
N
N
N
23
22
8b
Scheme 1. Reagents and conditions: (i) OsO4, NMMO, acetone–H2O, rt, overnight, 81%; (ii) mCPBA, CHCl3, rt, overnight, 73%.

M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
4377
N
N
N
Cl
Cl
Cl
N
Y
N
N
N
HO
X
i
ii
ii
N
N
N
N
N
N
X
31
32
37, X = OH, Y = H
33, X = O
iv
iii
39
, X = H, Y = F
35
, X = gem-F,F
N
N
N
N
Cl
Cl
Cl
N
i
Y
iv
N
N
N
HO
N
N
N
X
X
38, X = OH, Y = H
40
34, X = O
36
iii
, X = H, Y = F
, X = gem-F,F
Scheme 4. Reagents and conditions: (i) PDC, CHCl3, rt, overnight, 88% for 33, 92% for 34; (ii) NaBH4, MeOH, 0 °C, 1 h, 66% for 37, 63% for 38; (iii) DAST, CH2Cl2, pyridine, reflux,
30 h, 55% for 35, 45% for 36; (iv) DAST, CH2Cl2, pyridine, reflux, 12 h, 73% for 39, 63% for 40.
Table 4
N
N
Cl
Cl
Antiviral evaluation against CVB3 of the 9-substitutedpurines in Vero cells, all data
are mean values standard deviation for at least three independent experiments
N
N
N
i
N
N
37
N
N
Entry
Compound
EC50
(
lM)
EC90
(lM)
CC50 (lM)
1
2
3
4
5
6
7
8
9
7b
8b
9b
0.81 0.20
2.43 1.43
1.52 0.12
3.58 0.98
3.83 0.76
3.47 1.42
10.88 7.89
1.90 1.20
0.66 0.35
>50
2.98 2.07
5.72 0.69
1.27 0.50
1.00 0.01
>50
2.26 2.04
1.03 0.20
1.10 0.03
2.44 1.95
1.57 0.95
0.90 0.04
1.30 0.40
1.05 0.01
1.16 0.24
1.00 0.02
0.83 0.05
0.95 0.02
7.83 4.90
22.50 4.37
3.69 0.51
3.70 2.23
1.2 0.1
1.82 0.91
5.87 4.15
ND
8.49 4.32
9.43 4.72
36.26 26.57
>50
ND
>50
>50
ND
ND
ND
ND
>50
36.52 26.40
3.46 2.91
ND
ND
ND
>50
>50
43.85 8.70
>50
>50
>50
>50
43.35 9.40
>50
41
42
N
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22
23
31
32
33
34
37
38
35
Cl
N
Cl
i
N
N
38
N
N
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
>50
Scheme 5. Reagents and conditions: (i) 6-chloropurine, PPh3, DIAD, THF, rt 15 h to
reflux 10 h, 50% for 41, 55% for 42.
13.38 9.34
16.98 4.21
>50
>50
>50
>50
>50
47.85 3.05
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
N
Cl
N
NH2
i
i
N
N
N
N
N
NH2
1.78 0.89
ND
ND
34.40 27.97
ND
1.70 0.85
1.96 0.98
ND
43
N
Cl
45
36
39
40
41
42
45
46
TTP-8307
N
Cl
N
NH2
N
ND
8.81 4.48
ND
>50
N
49.76 0.35
19.07 1.67
>50
N
N
N
NH2
ND
44
N
Cl
46
rine nucleobase with appropriate alcohols and (ii) a built-up strat-
egy from appropriate amines. All compounds were evaluated for
antiviral activity against coxsackievirus B3. Most analogues
showed activity in the low micromolar range with minimal cyto-
toxicity to the cell line used in this study. Several compounds
showed comparable or slightly better activity than previously re-
ported derivatives. Studies on the mode of action of the most
promising compounds (7b and 15b) are ongoing.
Scheme 6. Reagents and conditions: (i) (1) 4,6-dichloropyrimidin-5-amine, Et3N,
EtOH, 100 °C, 10 days, (2) HC(OEt)3, HCl, 3 days, 19% for 45, 20% for 46.
compounds 17b and 18b bearing phenyl substituent either in posi-
tion
5 or 6 on the bicyclic scaffold showed considerable
cytotoxicity.
4. Conclusion
5. Experimental section
5.1. Chemistry
In summary, we have synthesized a novel class of coxsackievi-
rus inhibitors derived from a bicyclo[2.2.1]heptane scaffold. Two
methodologies were employed for introduction of the chloropurine
moiety to the scaffold: (i) the Mitsunobu reaction of the chloropu-
Melting points were determined on a Büchi B-540 apparatus.
NMR spectra (d, ppm; J, Hz) were measured on a Bruker Avance

4378
M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
II-600 and/or Bruker Avance II-500 instruments (600.1 or
500.0 MHz for 1H and 150.9 or 125.7 MHz for 13C) in hexadeuterat-
ed dimethyl sulfoxide and referenced to the solvent signal (d 2.50
and 39.70, respectively). Mass spectra were measured on a LTQ
Orbitrap XL (Thermo Fisher Scientific) using electrospray ioniza-
tion (ESI) and a GCT Premier (Waters) using EI. Column chromatog-
raphy was performed on Silica gel 60 (Fluka) and thin-layer
chromatography (TLC) on Silica gel 60 F254 foils (Merck). Solvents
were evaporated at 2 kPa and bath temperature 30–60 °C; the
compounds were dried at 13 Pa and 50 °C. The elemental analyses
were obtained on a Perkin–Elmer CHN Analyzer 2400, Series II Sys
(Perkin–Elmer). The elemental compositions for all compounds
agreed to within 0.4% of the calculated values. For all the tested
compounds satisfactory elemental analysis was obtained support-
ing >95% purity. Bicyclo[2.2.1]hept-5-en-2-yl acetate 24 (mixture
of endo/exo isomers) was purchased from Sigma–Aldrich.
1.7, 1.7(H-30), 2.51–2.47 (1H, m, H-40), 1.84 (1H, tdd, J 5.2, 5.2,
1.4, 1.0 Hz, H-20), 1.69–1.62 (1H, dm, J 10.6 Hz, H-50b), 1.52–1.50
(2H, m, H-10, H-60), 1.45–1.39 (1H, dm, J 10.6 Hz, H-50a), 1.29–
1.22 (1H, dm, J 11.4 Hz, H-70b), 1.10–1.02 (1H, dm, J 11.3 Hz, H-
70a). 13C NMR (125.73 MHz): 152.22 (C-4), 151.54 (C-2), 149.16
(C-6), 146.58 (C-8), 131.52 (C-5), 60.54 (C-30), 33.49 (C-40), 31.38
(C-50), 29.17 (C-70), 12.92 (C-20), 12.10 (C-60), 11.05 (C-10). ESI MS,
m/z (rel%): 247/249 (100/31) [M+H]. For C12H11ClN4 (246.7) calcd:
C, 58.42; H, 4.49; Cl, 14.37; N, 22.71; found: C, 58.27; H, 4.41; Cl,
14.23; N, 22.53.
*
*
*
5.1.1.4. 9-[(1R ,2S ,4R )-Bicyclo[2.2.1]hept-5-en-2-ylmethyl]-6-
chloro-9H-purine (10b). Yield 74%. Mobile phase: toluene–ethyl
acetate (4:1), viscous yellowish oil. 1H NMR (499.95 MHz): 8.79
(1H, s, H-8), 8.78 (1H, s, H-2), 6.08 (1H, dd, J 5.7, 2.9 Hz, H-50),
6.04 (1H, dd, J 5.7, 3.1 Hz, H-60), 4.38–4.29 (2H, m, CH2N), 2.87–
2.83 (1H, m, H-40), 2.55–2.51 (1H, m, H-10), 2.02–1.98 (1H, m, H-
20), 1.56–1.48 (1H, dm, J 8.6 Hz, H-70b), 1.35–1.29 (2H, m, H-30exo,
H-70a), 1.18–1.14 (1H, m, H-30endo). 13C NMR (125.73 MHz):
152.25 (C-4), 151.72 (C-2), 149.22 (C-6), 147.82 (C-8), 137.19 (C-
50), 136.26 (C-60), 131.00 (C-5), 48.83 (CH2N), 44.92 (C-70), 43.99
(C-10), 41.58 (C-40), 39.17 (C-20), 30.33 (C-30). ESI MS, m/z (rel%):
261/263 (100/33) [M+H]. For C13H13ClN4 (260.7) calcd: C, 59.89;
H, 5.03; Cl, 13.60; N, 21.49; found: C, 59.49; H, 5.08; Cl, 13.95; N,
21.15.
5.1.1. General method for the Mitsunobu coupling of a alcohol
with a nucleobase
To a mixture of the alcohol (0.50 g, 1 equiv), triphenylphosphine
(1.3 equiv), and 6-chloropurine (1.3 equiv) in THF (35 mL) a solu-
tion of the diisopropyl azadicarboxylate (1.3 equiv) in THF
(15 mL) was dropwise added. The resulting mixture was stirred
overnight (TLC control) and then eventually heated for reflux until
the starting alcohol was consumed (TLC control). The reaction mix-
ture was evaporated and the residue was chromatographed on sil-
ica gel column (200 g) in appropriate mobile phase. Finally, the
pure product was obtained after crystallization.
*
*
*
5.1.1.5. 9-[(1R ,2R ,4S )-Bicyclo[2.2.1]hept-2-ylmethyl]-6-chl-
oro-9H-purine (11b). Yield 68%. Mobile phase: toluene–ethyl
acetate (4:1), viscous yellowish oil. 1H NMR (499.95 MHz): 8.77
(1H, s, H-2), 8.76 (1H, s, H-8), 4.15 (1H, dd, J 13.9, 8.7 Hz, CH2N),
4.01 (1H, dd, J 13.9, 7.4 Hz, CH2N), 2.22–2.18 (1H, m, H-40), 2.11–
2.07 (1H, m, H-20), 1.93–1.89 (1H, m, H-10), 1.49–1.43 (1H, dm, J
9.8 Hz, H-70b), 1.42–1.38 (2H, m, H-50exo, H-60exo), 1.28 (1H, ddd,
J 13.2, 8.6, 2.3 Hz, H-30endo), 1.16–1.12 (1H, m, H-30exo), 1.14–
1.06 (1H, dm, J 9.8, H-70a), 1.07–1.03 (2H, m, H-50endo, H-60endo).
13C NMR (125.73 MHz): 152.26 (C-4), 151.72 (C-2), 149.22 (C-6),
147.85 (C-8), 130.93 (C-5), 48.37 (CH2N), 41.97 (C-20), 38.66 (C-
10), 36.18 (C-40), 34.99 (C-70), 34.71 (C-30), 29.23 (C-60), 28.46 (C-
50). ESI MS, m/z (rel%): 263/265 (100/33) [M+H]. For C13H15ClN4
(262.7) calcd: C, 59.43; H, 5.75; Cl, 13.49; N, 21.32; found: C,
59.25; H, 5.90; Cl, 13.30; N, 21.05.
*
*
*
5.1.1.1. 9-[(1R ,2R ,4S )-Bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-
purine (7b). Yield 41%. Mobile phase: toluene–ethyl acetate
(6:1?4:1). Mp 104–105 °C (water–methanol, white crystals). 1H
NMR (600.13 MHz): 8.85 (1H, s, H-8), 8.77 (1H, s, H-2), 4.56 (1H,
ddd, J 8.4, 4.5, 0.9 Hz, H-20), 2.55 (1H, bd, J 4.6 Hz, H-10), 2.43
(1H, bt, J 4.2, 4.2 Hz, H-40), 2.09–2.05 (1H, m, H-30endo), 2.00 (1H,
ddd, J 13.4, 8.5, 2.3 Hz, H-30exo), 1.71 (1H, dm, J 10.3 Hz, H-70a),
1.62 (1H, tt, J 12.2, 12.2, 4.5, 4.5 Hz, H-60endo), 1.56–1.54 (1H, m,
H-50endo), 1.40 (1H, dddd, J 12.1, 9.1, 4.1, 2.3 Hz, H-60exo), 1.29–
1.27 (1H, m, H-70b), 1.29–1.25 (1H, m, H-50exo). 13C NMR
(150.92 MHz): 152.11 (C-4), 151.45 (C-2), 149.11 (C-6), 145.62
(C-8), 131.43 (C-5), 58.37 (C-20), 42.16 (C-10), 37.76 (C-30), 35.93
(C-40), 35.82 (C-70), 27.95 (C-50), 26.94 (C-60). ESI MS, m/z (rel%):
249/251 (100/35) [M+H]. For C12H13ClN4 (248.7) calcd: C, 57.95;
H, 5.27; Cl, 14.25; N, 22.53; found: C, 57.93; H, 5.32; Cl, 14.29; N,
22.34.
*
*
*
5.1.1.6. 6-Chloro-9-[(1R ,2R ,4R )-1,7,7-trimethylbicyclo[2.2.1]
hept-2-yl]-9H-purine (12b). Yield 35%. Mobile phase: toluene–
ethyl acetate (5:1). Mp 132.3–134.5 °C (water–methanol, white
crystals). 1H NMR (600.13 MHz): 9.00 (1H, s, H-8), 8.79 (1H, s, H-
2), 4.70 (1H, dd, J 9.5, 6.9 Hz, H-20), 2.77 (1H, dddd, J 13.3, 6.9,
4.0, 3.2 Hz, H-30exo), 1.99 (1H, dd, J 13.2, 9.6 Hz, H-30endo), 1.95
(1H, t, J 4.2, 4.2 Hz, H-40), 1.84–1.80 (1H, m, H-50exo), 1.66 (1H,
td, J 12.2, 12.2, 4.9 Hz, H-60exo), 1.45 (1H, ddd, J 12.4, 9.2, 3.6 Hz,
H-60endo), 1.33 (1H, ddd, J 12.3, 9.3, 4.9 Hz, H-50endo), 0.99 (3H,
s, 70-CH3), 0.85 (3H, s, 70-CH3), 0.65 (3H, s, 10-CH3). 13C NMR
(150.92 MHz): 153.48 (C-4), 151.41 (C-2), 149.28 (C-6), 146.77
(C-8), 131.18 (C-5), 63.15 (C-20), 50.66 (C-10), 47.24 (C-70), 44.59
(C-40), 36.64 (C-60), 34.31 (C-30), 26.55 (C-50), 21.13 and 19.82
(2 ꢀ 70-CH3), 12.06 (10-CH3). For C15H19ClN4 (290.8) calcd: C,
61.96; H, 6.59; Cl, 12.19; N, 19.27; found: C, 61.89; H, 6.70; Cl,
12.31; N, 19.07.
*
*
*
5.1.1.2. 9-[(1R ,2S ,4R )-Bicyclo[2.2.1]hept-5-en-2-yl]-6-chloro-
9H-purine (8b). Yield 62%. Mobile phase: toluene–ethyl acetate
(6:1?4:1). Mp 117.5–118.5 °C (water–methanol, white crystals).
1H NMR (600.13 MHz): 8.93 (1H, s, H-8), 8.78 (1H, s, H-2), 6.37
(1H, dd, J 5.7, 2.9 Hz, H-50), 6.26 (1H, dd, J 5.7, 3.2 Hz, H-60), 4.46
(1H, ddd, J 8.3, 3.9, 1.6 Hz, H-20), 3.23–3.19 (1H, m, H-10), 3.07–
3.04 (1H, m, H-40), 2.18 (1H, dt, J 12.6, 3.7, 3.7 Hz, H-30exo), 1.88
(1H, ddd, J 12.6, 8.3, 2.6 Hz, H-30endo), 1.82–1.76 (1H, dm, J
9.2 Hz, H-70b), 1.59–1.52 (1H, dm, J 9.2 Hz, H-70a). 13C NMR
(150.92 MHz): 152.60 (C-4), 151.49 (C-2), 149.14 (C-6), 145.96
(C-8), 140.21 (C-50), 134.48 (C-60), 131.51 (C-5), 55.82 (C-20),
47.55 (C-10), 46.23 (C-70), 41.28 (C-40), 32.14 (C-30). ESI MS, m/z
(rel%): 343 (100), 269/271(90/22) [M+Na], 247/249 (38/12)
[M+H]. For C12H11ClN4 (246.7) calcd: C, 58.42; H, 4.49; Cl, 14.37;
N, 22.71; found: C, 58.44; H, 4.38; Cl, 14.34; N, 22.44.
* * *
6-Chloro-9-[(1R ,2S ,4S )-3,3-dimethylbicyclo[2.2.1]
5.1.1.7.
hept-2-yl]-9H-purine (13b). Yield 35%. Mobile phase: toluene–
ethyl acetate (5:1). Mp 112.3–114 °C (water–methanol, white crys-
tals). 1H NMR (600.13 MHz): 8.78 (1H, s, H-8), 8.78 (1H, s, H-2).
4.15 (1H, d, J 1.6 Hz, H-20), 2.70 (1H, dm, J 4.8 Hz, H-10), 2.28 (1H,
dm, J 10.8 Hz, H-70b), 1.92 (1H, dm, J 3.6 Hz, H-40), 1.76–1.72 (2H,
m, H-50endo, H-60exo), 1.48–1.40 (3H, m, H-50exo, H-60endo, H-
2,6
*
*
*
5.1.1.3. 6-Chloro-9-[(1R ,3S ,4S )-tricyclo[2.2.1.0 ]hept-3-yl]-
9H-purine (9b). Yield 14%. Mobile phase: toluene–ethyl acetate
(4:1). Mp 92–93 °C (hexanes–ether, white crystals). 1H NMR
(499.95 MHz): 8.77 (1H, s, H-2), 8.65 (1H, s, H-8), 4.42 (1H, t, J

M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
4379
70a), 1.27 (3H, s, 30-CH3), 0.39 (3H, s, 30-CH3). 13C NMR
(150.92 MHz): 152.86 (C-4), 151.82 (C-2), 149.22 (C-6), 145.68
(C-8), 131.57 (C-5), 68.08 (C-20), 48.03 (C-40), 44.46 (C-30), 42.68
(C-10), 37.09 (C-70), 28.22 (C-60), 26.14 (30-CH3), 23.59 (C-50),
23.37 (30-CH3). ESI MS, m/z (rel%): 277/279 (100/45) [M+H]. For
C14H17ClN4 (276.8) calcd: C, 60.76; H, 6.19; Cl, 12.81; N, 20.24;
found: C, 60.48; H, 6.36; Cl, 12.57; N, 19.99.
4.2 Hz, H-20), 3.04 (1H, dd, J 9.0, 5.5 Hz, H-60), 2.76 (1H, bs, H-10),
2.57–2.53 (1H, m, H-40), 2.19–2.08 (2H, m, H-30), 1.89 (1H, ddd, J
12.3, 8.9, 1.9 Hz, H-50endo), 1.71–1.64 (2H, m, H-50exo, H-70),
1.60–1.54 (1H, dm, J 10.7 Hz, H-70a). 13C NMR (125.73 MHz):
152.25 (C-4), 151.51 (C-2), 149.13 (C-6), 145.74 (C-8), 145.59 (C-
100), 131.54 (C-5), 128.62 (C-300), 127.28 (C-200), 126.06 (C-400),
58.96 (C-20), 48.21 (C-10), 44.20 (C-60), 37.47 (C-50), 36.90 (C-30),
36.42 (C-40), 33.82 (C-70). ESI MS, m/z (rel%): 325/327 (100/33)
[M+H]. For C18H17ClN4 (324.8) calcd: C, 66.56; H, 5.28; Cl, 10.92;
N, 17.25; found: C, 66.29; H, 5.30; Cl, 10.88; N, 16.99.
2,6
*
*
*
*
*
5.1.1.8.
6-Chloro-9-[(1R ,2R ,6R ,7R ,8S )-tricyclo[5.2.1.0
]dec-8-yl]-9H-purine (14b). Yield 46%. Mobile phase: toluene–
ethyl acetate (7:1). Mp 104.5–106.5 °C (water–acetone, white
powder). 1H NMR (600.13 MHz): 8.86 (1H, s, H-8), 8.77 (1H, s, H-
2), 4.48 (1H, ddd, J 8.4, 4.2, 1.0 Hz, H-80), 2.33 (1H, s, H-70), 2.15
(1H, d, J 4.4 Hz, H-10), 2.05 (1H, dt, J 13.3, 4.3, 4.3 Hz, H-90exo),
2.04–2.01 (1H, m, H-60), 1.95–1.91 (1H, m, H-90endo), 1.93–1.89
(1H, m, H-20), 1.90–1.83 (2H, m, H-30b, H-50b), 1.67–1.63 (1H, m,
H-40), 1.51–1.53 (1H, dm, J 11.1 Hz, H-100b), 1.48–1.40 (1H, dm, J
11.1 Hz, H-100a), 1.26–1.17 (2H, m, H-40), 1.01–0.93 (2H, m, H-
30a, H-50a). 13C NMR (150.92 MHz): 152.25 (C-4), 151.47 (C-2),
149.12 (C-6), 145.67 (C-8), 131.48 (C-5), 57.90 (C-80), 46.93 (C-
20), 46.40 (C-70), 45.42 (C-60), 40.21 (C-10), 36.81 (C-90), 31.87 (C-
50), 31.48 (C-30), 29.95 (C-100), 27.46 (C-40). ESI MS, m/z (rel%):
289/291 (100/29) [M+H]. For C15H17ClN4 (288.8) calcd: C, 62.39;
H, 5.93; Cl, 12.28; N, 19.40; found: C, 62.25; H, 5.86; Cl, 12.53; N,
19.17.
* * * *
6-Chloro-9-[(1R ,2S ,4R ,5S )-5-phenylbicyclo[2.2.1]
5.1.1.12.
hept-2-yl]-9H-purine (18b). Yield 75%. Mobile phase: toluene–
ethyl acetate (5:1). Mp 129–131 °C (ether, white powder). 1H
NMR (499.95 MHz): 8.90 (1H, s, H-8), 8.79 (1H, s, H-2), 7.32–7.25
(4H, m, H-200, H-300), 7.20–7.15 (1H, m, H-400), 4.70 (1H, bdd, J 8.2,
4.9 Hz, H-20), 2.91 (1H, dd, J 8.9, 5.6 Hz, H-50), 2.68 (1H, bd, J
4.6 Hz, H-10), 2.52–2.48 (1H, m, H-40), 2.25–2.15 (2H, m, H-30),
2.00 (1H, ddd, J 12.9, 9.0, 2.4 Hz, H-60endo), 1.77 (1H, dt, J 12.9,
5.1, 5.1 Hz, H-60exo), 1.75–1.72 (1H, m, H-70b), 1.60–1.54 (1H,
dm, J 10.7 Hz, H-70a). 13C NMR (125.73 MHz): 152.23 (C-4),
151.52 (C-2), 149.19 (C-6), 146.19 (C-100), 145.69 (C-8), 131.51
(C-5), 128.60 (C-300), 127.17 (C-200), 125.99 (C-400), 58.02 (C-20),
45.42 (C-50), 42.87 and 42.85 (C-10 and C-40), 38.21 (C-30), 35.71
(C-60), 33.59 (C-70). ESI MS, m/z (rel%): 325/327 (100/33) [M+H],
329 (65). For C18H17ClN4 (324.8) calcd: C, 66.56; H, 5.28; Cl,
10.92; N, 17.25; found: C, 66.38; H, 5.20; Cl, 11.15; N, 16.94.
2,7
*
*
*
5.1.1.9. 6-Chloro-9-[(1R ,8R ,9S )-tricyclo[6.2.1.0
]undeca-
2,4,6-trien-9-yl]-9H-purine (15b). Yield 78%. Mobile phase: tolu-
ene–ethyl acetate (8:1?6:1). Mp 202–203.5 °C (water–methanol,
white crystals). 1H NMR (499.84 MHz): 9.03 (1H, s, H-8), 8.80
(1H, s, H-2), 7.37–7.33 (1H, m, H-60), 7.30–7.26 (1H, m, H-30),
7.17–7.12 (2H, m, H-40, H-50), 4.53 (1H, ddd, J 8.3, 4.2, 1.0 Hz, H-
90), 3.73 (1H, bs, H-80), 3.58–3.54 (1H, m, H-10), 2.52 (1H, dt, J
12.9, 4.0, 4.0 Hz, H-100exo), 2.20 (1H, dt, J 9.8, 1.4, 1.4 Hz, H-110b),
1.99 (1H, ddd, J 12.9, 8.4, 2.5 Hz, H-100endo), 1.88 (1H, d, J 9.7 Hz,
H-110a). 13C NMR (125.70 MHz): 152.52 (C-4), 151.63 (C-2),
149.23 (C-6), 148.51 (C-20), 146.07 (C-8), 144.91 (C-70), 131.56
(C-5), 126.95 and 126.22 (C-40 and C-50), 122.10 (C-60), 121.31 (C-
30), 57.16 (C-90), 49.36 (C-80), 46.90 (C-110), 43.12 (C-10), 34.91 (C-
5.1.2. General method for the nucleobase built-up from amines
A mixture of amine (3 mmol), 4,6-dichloropyrimidin-5-amine
(4.5 mmol), and triethylamine (1.8 mL) in ethanol (9 mL) was
heated in a pressure vessel at 105 °C for 6 days and, after cooling,
was evaporated. The residue was chromatographed on a column
of silica gel (200 g). The pyrimidine intermediate was eluted with
appropriate mobile phase and this intermediate was immediately
used in the next step. Concentrated hydrochloric acid (1 mL) was
added to a suspension of pyrimidine intermediate in triethyl ortho-
formate (80 mL) and the reaction mixture was vigorously stirred
for 3 days at room temperature. Solution was evaporated and the
residue was crystallized from water–methanol (95:5) to afford
product.
100). ESI MS, m/z (rel%): 297/299 (100/30) [M+H]. For
1
C16H13ClN4 ꢀ H2O (301.3) calcd: C, 64.76; H, 4.42; Cl, 11.95; N,
4
18.88; found: C, 64.89; H, 4.21; Cl, 12.19; N, 18.53.
3,7
*
*
*
*
*
5.1.2.1. 6-Chloro-9-[(1R ,2S ,3S ,6S ,7R )-4-oxatricyclo[4.2.1.0
]non-2-yl]-9H-purine (19b). Yield 71%. Mobile phase: toluene–
ethyl acetate (5:1?1:1). Mp 166–167 °C (water–methanol). 1H
NMR (600.13 MHz): 8.78 (1H, s, H-8), 8.77 (1H, s, H-2), 4.76 (1H,
dd, J 5.0, 1.4 Hz, H-30), 4.14 (1H, d, J 2.1 Hz, H-20), 3.80 (1H, dd, J
8.0, 4.2 Hz, H-50b), 3.75 (1H, d, J 8.0 Hz, H-50a), 2.76–2.72 (1H, m,
H-70), 2.62–2.58 (1H, m, H-10), 2.50–2.46 (1H, m, H-60), 2.06 (1H,
ddd, J 12.8, 10.8, 4.5 Hz, H-90exo), 1.88–1.83 (1H, dm, J 11.3 Hz,
H-80b), 1.72–1.65 (1H, dm, J 11.4 Hz, H-80a), 1.29 (1H, dt, J 12.8,
2.3 Hz, H-90endo). 13C NMR (150.92 MHz): 152.30 (C-4), 151.57
(C-2), 149.23 (C-6), 145.79 (C-8), 131.30 (C-5), 83.92 (C-30), 74.51
(C-50), 66.85 (C-20), 45.78 (C-70), 39.03 (C-10), 37.16 (C-60), 35.45
(C-80), 35.13 (C-90). ESI MS, m/z (rel%): 299/301 (100/35) [M+Na],
277/279 (73/23) [M+H]. For C13H13ClN4O (276.7) calcd: C, 56.42;
H, 4.74; Cl, 12.81; N, 20.25; found: C, 56.21; H, 4.56; Cl, 13.08; N,
19.96.
*
*
*
5.1.1.10. 6-Chloro-9-[(1R ,2S ,4R )-1,2,3,4-tetrahydroanthracen-
2-yl]-9H-purine (16b). Yield 65%. Mobile phase: toluene–ethyl
acetate (10:1). Mp 267–268.5 °C (methanol, white powder, decom-
position). 1H NMR (499.84 MHz): 9.08 (1H, s, H-8), 8.83 (1H, s, H-
2), 7.84 (1H, s, H-90), 7.82–7.92 (2H, m, H-50, H-80), 7.74 (1H, s, H-
100), 7.48–7.44 (2H, m, H-60, H-70), 4.71–4.65 (1H, m, H-20), 3.90–
3.85 (1H, m, H-10), 3.73–3.69 (1H, m, H-40), 2.66 (1H, dt, J 13.0,
4.2, 4.2 Hz, H-30exo), 2.36–2.30 (1H, dm, J 9.9 Hz, H-110b), 2.12
(1H, ddd, J 13.0, 8.6, 2.3 Hz, H-30endo), 1.99–1.92 (1H, dm, J
9.9 Hz, H-110a). 13C NMR (125.72 MHz): 152.51 (C-4), 151.67 (C-
2), 149.27 (C-6), 146.58 (C-40a), 146.10 (C-8), 143.43 (C-90a),
132.96 (C-100a), 132.54 (C-80a), 131.62 (C-5), 128.07 and 127.89
(C-50 and C-80), 125.75 and 125.47 (C-60 and C-70), 120.17 (C-90),
118.98 (C-100), 57.38 (C-20), 49.21 (C-10), 45.82 (C-110), 42.90 (C-
40), 35.45 (C-30). ESI MS, m/z (rel%): 369/371 (100/33) [M+Na].
For C20H15ClN4 (346.8) calcd: C, 69.26; H, 4.36; Cl, 10.22; N,
16.15; found: C, 69.22; H, 4.51; Cl, 10.37; N, 15.75.
3,7
*
*
*
*
*
5.1.2.2. 6-Chloro-9-[(1R ,3R ,6R ,7R ,9R )-4-oxatricyclo[4.2.1.0
]
non-9-yl]-9H-purine (20b). Yield 65%. Mobile phase: toluene–
ethyl acetate (5:1?1:1). Mp 196–198 °C (water–methanol, white
crystals). 1H NMR (600.13 MHz): 8.88 (1H, s, H-8), 8.79 (1H, s,
H-2), 4.37 (1H, dd, J 7.7, 4.9 Hz, H-30), 4.33 (1H, m, H-90), 3.94
(1H, d, J 8.5 Hz, H-50a), 3.80 (1H, dd, J 8.5, 4.6 Hz, H-50b), 3.04
(1H, bt, J 5.2, 5.2 Hz, H-60), 2.74 (1H, tq, J 4.8, 4.8, 1.4, 1.4, 1.4 Hz,
*
*
*
*
5.1.1.11. 6-Chloro-9-[(1R ,2S ,4R ,6R )-6-phenylbicyclo[2.2.1]
hept-2-yl]-9H-purine (17b). Yield 65%. Mobile phase: toluene–
ethyl acetate (5:1). Mp 102–103.5 °C (ether, white powder). 1H
NMR (499.95 MHz): 8.91 (1H, s, H-8), 8.78 (1H, s, H-2), 7.31–7.26
(4H, m, H-200, H-300), 7.19–7.15 (1H, m, H-400), 4.77 (1H, dd, J 8.0,

4380
M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
H-70), 2.58 (1H, bd, J 3.9 Hz, H-10), 1.99–1.93 (1H, dm, J 11.2 Hz, H-
80b), 1.73 (1H, ddd, J 13.3, 7.7, 4.0 Hz, H-20exo), 1.54–1.47 (1H, dm, J
11.2 Hz, H-80a), 1.22 (1H, dd, J 13.3, 3.3 Hz, H-20endo). 13C NMR
(150.92 MHz): 152.22 (C-4), 151.57 (C-2), 149.17 (C-6), 145.72
(C-8), 131.49 (C-5), 77.84 (C-30), 71.58 (C-50), 65.78 (C-90), 46.70
(C-60), 45.76 (C-70), 39.50 (C-10), 38.16 (C-20), 35.18 (C-80). ESI MS,
m/z (rel%): 277/279 (100/20) [M+H]. For C13H13ClN4O (276.7)
calcd: C, 56.42; H, 4.74; Cl, 12.81; N, 20.25; found: C, 56.31; H,
4.75; Cl, 12.72; N, 20.04.
263/265 (100/31) [M+H]. For C12H11ClN4O (262.7) calcd: C,
54.87; H, 4.22; Cl, 13.50; N, 21.33; found: C, 54.63; H, 4.07; Cl,
13.79; N, 20.59.
*
*
*
5.1.5. (1R ,2R ,4S )-6-Oxobicyclo[2.2.1]hept-2-yl benzoate (25)
*
*
*
and (1R ,2S ,4R )-5-oxobicyclo[2.2.1]hept-2-yl benzoate (26)
To a mixture of bicyclo[2.2.1]hept-5-en-2-yl acetate 24 (mix-
ture of endo/exo isomers, 11.27 g, 74 mmol) in dry THF (15 mL)
BH3–THF complex (1 M solution in THF, 35 mL) at 0 °C was drop-
wise added. The reaction mixture was stirred under argon for 2
hours at 0 °C. Next, a few drops of water were carefully added fol-
lowed by a suspension of NaBO3 ꢀ 4 H2O (33 g, 215 mmol) in
water (70 mL). The reaction mixture was vigorously stirred over-
night at room temperature. Inorganic salts were removed by filtra-
tion and the filtrate was extracted with ether (4 ꢀ 150 mL). The
organic phase was dried over anhydrous sodium sulfate and evap-
orated (9.55 g). Benzoyl chloride (8 mL, 69 mmol) was added to a
stirred and cooled solution hydroborated intermediate in pyridine
(100 mL) and the mixture was left at room temperature overnight.
Water (3 mL) was then added and, after 15 min, the solvent was
evaporated. The residue was partitioned between ethyl acetate
(250 mL) and water (200 mL). The organic phase was washed with
water (150 mL), 5% hydrochloric acid (120 mL), saturated aqueous
NaHCO3 (3 ꢀ 150 mL), dried over anhydrous sodium sulfate, and
the solvent was evaporated (15.12 g of benzoylated intermediate).
This crude intermediate was dissolved in methanol (300 mL) and
to this solution K2CO3 (2 g, 14.5 mmol). The reaction mixture was
stirred for 1.5 hour, neutralized with 5% solution of hydrochloric
acid and evaporated. The residue was partitioned between ethyl
acetate (300 mL) and water (100 mL). The organic phase was
washed with water (100 mL), dried over anhydrous sodium sulfate
and evaporated (12.47 g of deacetylated intermediate). A mixture
of pyridinium dichromate (30 g, 79.7 mmol), molecular sieves
(3A, powdered, 30 g) and dichloromethane (250 mL) was stirred
at room temperature for 15 min. A solution of the deacetylated
intermediate (12.47 g) in dichloromethane (100 mL) was added
to the mixture. After 15 h stirring at room temperature, the mix-
ture was filtered and the filtrates were evaporated. The residue
was stirred with ether, the mixture was filtered with a Celite pad
and the filtrates were taken down. Chromatography of the residue
on silica gel (400 g) in toluene–ethyl acetate (30:1) afforded 5.09 g
(30%) of ketobenzoate 25 and 2.76 g (16%) of ketobenzoate 26, both
as colorless oils.
*
*
*
5.1.2.3.
9-[(1R ,2S ,4S )-Bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-
purine (21b). Yield 40%. Mobile phase: toluene–ethyl acetate
(5:1?2:1). Mp 99–100.5 °C (water–methanol, white crystals). 1H
NMR (499.84 MHz): 8.87 (1H, s, H-8), 8.76 (1H, s, H-2), 4.96–4.92
(1H, m, H-20), 2.74–2.72 (1H, m, H-10), 2.40–2.36 (1H, m, H-40),
2.20–2.18 (1H, m, H-30exo), 1.98 (1H, ddd, J 13.3, 5.0, 3.0 Hz, H-
30endo), 1.69–1.62 (1H, dm, J 9.9 Hz, H-70b), 1.59–1.53 (2H, m, H-
50), 1.46–1.40 (1H, dm, J 9.9 Hz, H-70a), 1.34–1.31 (1H, m, H-60exo),
0.94–0.91 (1H, m, H-60endo). 13C NMR (125.70 MHz): 152.81 (C-4),
151.52 (C-2), 149.22 (C-6), 146.50 (C-8), 131.53 (C-5), 57.11 (C-20),
41.11 (C-10), 37.88 (C-70), 36.41 (C-40), 32.38 (C-30), 28.61 (C-50),
21.79 (C-60). ESI MS, m/z (rel%): 249/251 (100/33) [M+H]. For
C12H13ClN4 (248.7) calcd: C, 57.95; H, 5.27; Cl, 14.25; N, 22.53;
found: C, 57.88; H, 5.32; Cl, 14.45; N, 22.21.
*
*
*
*
*
5.1.3. (1R ,2R ,3S ,4S ,5S )-5-(6-Chloro-9H-purin-9-yl)bicyclo-
[2.2.1]heptane-2,3-diol (22)
Unsaturated derivative 8b (250 mg, 1.01 mmol) was dissolved
in mixture of water–acetone (15 mL, 1:1). Solution of NMMO
(50% in water, 2 mL) and water solution of osmium tetroxide
(50 lL, 100 mg/5 mL) was added and the reaction mixture was stir-
red at rt overnight. The reaction mixture was evaporated and the
residue was chromatographed on silica gel column (100 g) in ethyl
acetate–acetone–ethanol–water (225:15:6:4). It was obtained
230 mg (81%) of the product 22. Mp 198–200 °C (water, white
1
powder). H NMR (600.13 MHz): 8.84 (1H, s, H-80), 8.76 (1H, s,
H-20), 4.82 (1H, d, J 6.0 Hz, 3-OH), 4.80 (1H, d, J 6.1 Hz, 2-OH),
4.49 (1H, dd, J 8.5, 4.3 Hz, H-6), 3.80–3.76 (1H, m, H-2), 3.68–
3.64 (1H, m, H-3), 2.40 (1H, bs, H-1), 2.22–2.16 (1H, dm, J 4.7 Hz,
H-4), 2.01–1.97 (1H, m, H-5exo), 1.89 (1H, ddd, J 13.8, 8.5, 2.2 Hz,
H-5endo), 1.83–1.77 (1H, dm, J 10.8 Hz, H-7b), 1.57–1.51 (1H,
dm, J 10.8 Hz, H-7a). 13C NMR (150.92 MHz): 152.15 (C-40),
151.45 (C-20), 149.13 (C-60), 145.67 (C-80), 131.48 (C-50), 72.51 (C-
3), 71.28 (C-2), 54.61 (C-6), 49.10 (C-1), 42.77 (C-4), 32.96 (C-5),
29.70 (C-7). ESI MS, m/z (rel%): 281/283 (100/30) [M+H]. For
C12H13ClN4O2 ꢀ ½ H2O (289.7) calcd: C, 49.57; H, 4.87; Cl, 12.24;
N, 19.34; found: C, 49.82; H, 4.55; Cl, 12.51; N, 19.19.
* * *
(1R ,2R ,4S )-6-Oxobicyclo[2.2.1]hept-2-yl benzoate
5.1.5.1.
1
(25). H NMR (499.95 MHz): 7.99–7.93 (2H, m, H-20), 7.68–7.64
(1H, m, H-40), 7.49–7.55 m, 2H (H-30), 5.03–4.96 (1H, dm, J
7.1 Hz, H-2), 2.80–2.76 (2H, m, H-1, H-4), 2.14–2.06 (1H, dm, J
17.8 Hz, H-5exo), 2.13 (1H, ddd, J 13.8, 7.2, 2.4 Hz, H-3endo),
2.02–1.96 (1H, dm, J 10.5 Hz, H-7b), 1.88–1.81 (3H, m, H-3exo, H-
5endo, H-7a). 13C NMR (125.73 MHz): 214.52 (C-6), 165.33 (COO),
133.70 (C-40), 129.81 (C-10), 129.43 (C-20), 128.97 (C-30), 72.56 (C-
2), 56.69 (C-1), 44.17 (C-5), 37.65 (C-3), 35.01 (C-7), 34.75 (C-4).
EI MS, m/z (rel%): 230 (11) [M], 202 (52), 105 (100) [Bz], 77 (55).
For C14H13O3 (230.3) calcd: C, 73.03; H, 6.13; found: C, 72.69; H,
6.02.
2,4
*
*
*
*
*
5.1.4. 6-Chloro-9-[(1R ,2R ,4S ,5S ,6S )-3-oxatricyclo[3.2.1.0
oct-6-yl]-9H-purine (23)
]
To a solution of unsaturated derivative 8b (250 mg, 1.01 mmol)
in dichloromethane (45 mL) was added m-chloroperbenzoic acid
(400 mg, 1.5 mmol, 65%) and the solution was stirred at rt over-
night. Reaction mixture was washed with saturated aqueous
Na2S2O3 (20 mL), saturated NaHCO3 (3 ꢀ 20 mL), dried over anhy-
drous sodium sulfate and evaporated. Residue was crystallized
from water–methanol to afford 193 mg (73%) of the product 23
as white crystals. Mp 159–163 °C (water–methanol). 1H NMR
(600.13 MHz): 8.88 (1H, s, H-8), 8.79 (1H, s, H-2), 4.60 (1H, ddd, J
8.0, 4.5, 1.5 Hz, H-60), 3.38 (1H, dd, J 3.8, 1.7 Hz, H-20), 3.34 (1H,
dd, J 3.7, 1.4 Hz, H-30), 2.92–2.86 (1H, m, H-10), 2.64–2.60 (1H, m,
H-40), 2.14–2.06 (2H, m, H-50endo, H-50exo), 1.38–1.31 (1H, dm, J
10.6 Hz, H-70b), 1.32–1.24 (1H, dm, J 10.7 Hz, H-70a). 13C NMR
(150.92 MHz): 152.20 (C-4), 151.53 (C-2), 149.18 (C-6), 145.96
(C-8), 131.38 (C-5), 54.21 (C-60), 50.83 (C-30), 48.91 (C-20), 42.68
(C-10), 36.88 (C-40), 33.84 (C-50), 23.95 (C-70). ESI MS, m/z (rel%):
* * *
(1R ,2S ,4R )-5-Oxobicyclo[2.2.1]hept-2-yl benzoate
5.1.5.2.
1
(26). H NMR (499.95 MHz): 8.02–7.96 (2H, m, H-20), 7.68–7.64
(1H, m, H-40), 7.56–7.50 (2H, m, H-30), 5.09–5.03 (1H, dm, J
6.9 Hz, H-2), 2.79–2.74 (1H, dm, J 5.2 Hz, H-1), 2.56–2.50 (1H,
dm, J 4.9 Hz, H-4), 2.13 (1H, ddd, J 17.7, 5.2, 1.2 Hz, H-6exo), 2.03
(1H, ddd, J 14.3, 6.9, 2.4 Hz, H-3endo), 1.96–1.89 (2H, m, H-6endo,
H-7b), 1.85 (1H, dddd, J 14.3, 5.0, 2.5, 1.4 Hz, H-3exo), 1.81–1.75
(1H, dm, J 10.4 Hz, H-7a). 13C NMR (125.73 MHz): 215.00 (C-6),
165.47 (COO), 133.62 (C-40), 129.98 (C-10), 129.41 (C-20), 128.97

M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
4381
(C-30), 75.50 (C-2), 48.46 (C-4), 40.63 (C-1), 39.40 (C-6), 34.12 (C-7),
33.22 (C-3). EI MS, m/z (rel%): 230 (31) [M], 212 (15), 108 (90), 105
(92) [Bz], 77 (100), 66 (93). For C14H13O3 (230.3) calcd: C, 73.03; H,
6.13; found: C, 72.91; H, 6.09.
(499.95 MHz): 8.88 (1H, s, H-80), 8.80 (1H, s, H-20), 7.97–7.93
(2H, m, H-200), 7.67–7.63 (1H, m, H-400), 7.55–7.49 (2H, m, H-300),
5.14–5.10 (1H, dm, J 7.0 Hz, H-2), 4.73 (1H, bt, J 6.5, 6.5 Hz, H-6),
2.86 (1H, bs, H-1), 2.58–2.55 (1H, m, H-4), 2.05–2.07 (2H, m, H-
5), 2.00 (1H, ddd, J 13.7, 7.1, 2.3 Hz, H-3endo), 1.90–1.86 (1H, dm,
J 10.8 Hz, H-7b), 1.80–1.72 (1H, dm, J 10.8 Hz, H-7a), 1.65–1.60
(1H, dm, J 13.7 Hz, H-3exo). 13C NMR (125.73 MHz): 165.38
(COO), 152.15 (C-40), 151.53 (C-20), 149.20 (C-60), 145.77 (C-80),
133.60 (C-400), 131.49 (C-50), 129.99 (C-100), 129.34 (C-200), 128.96
(C-300), 75.42 (C-2), 54.33 (C-6), 47.52 (C-1), 38.26 (C-3), 36.82 (C-
5), 35.19 (C-4), 33.57 (C-7). ESI MS, m/z (rel%): 369/371 (100/33)
[M+H]. For C19H17ClN4O2 (368.8) calcd: C, 61.87; H, 4.65; Cl,
9.61; N, 15.19; found: C, 61.54; H, 4.61; Cl, 9.49; N, 14.82.
*
*
*
*
*
*
5.1.6. (1R ,2S ,4S ,6S )-6-Hydroxybicyclo[2.2.1]hept-2-yl benzo-
*
*
ate (27) and (1R ,2S ,4R ,5R )-5-hydroxybicyclo[2.2.1]hept-2-yl
benzoate (28)
Ketobenzoate 25 or 26 (5.17 g, 22.45 mmol) was dissolved in
methanol (210 mL) and the solution was immersed in an ice-bath.
Sodium borohydride (517 mg, 13.7 mmol) was added in small por-
tions during 20 min. The reaction mixture was stirred at 0 °C for
1 h and evaporated, co-distilled with methanol (50 mL) and the
residue was partitionated between ethyl acetate (200 mL) and
brine (100 mL). The organic phase was washed with brine
(100 mL), dried over anhydrous sodium sulfate and evaporated.
Residue was chromatographed on a silica gel (350 g) in toluene–
ethyl acetate (7:1) to afford alcohol.
*
*
*
*
5.1.7.2. (1R ,2S ,4R ,5S )-5-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
hept-2-yl benzoate (30). Yield 3.44 g (contaminated with hydra-
zine byproduct), product crystallized water–ethanol (1:1) to afford
pure compound as white crystals, 1.92 g (48%). Mp 166–168 °C
1
(water–ethanol (1:1)). H NMR (499.95 MHz): 8.90 (1H, s, H-80),
*
*
*
*
5.1.6.1. (1R ,2S ,4S ,6S )-6-Hydroxybicyclo[2.2.1]hept-2-yl ben-
zoate (27). Yield 5.05 g (95%), colorless oil. 1H NMR
(499.95 MHz): 7.97–7.92 (1H, m, H-20), 7.67–7.63 (1H, m, H-40),
7.53–7.49 (2H, m, H-30), 5.43–5.39 (1H, m, H-2), 4.88 (1H, d, J
3.8 Hz, OH), 4.14–4.10 (1H, m, H-6), 2.41 (1H, d, J 4.8 Hz, H-1),
2.22 (1H, bt, J 4.6, 4.6 Hz, H-4), 1.95 (1H, ddd, J 13.3, 7.1, 2.4 Hz,
H-3endo), 1.82 (1H, dddd, J 12.6, 10.2, 4.6, 3.0 Hz, H-5exo), 1.62–
1.54 (1H, dm, J 10.2 Hz, H-7b), 1.55–1.51 (1H, m, H-3exo), 1.33–
1.27 (1H, dm, J 10.1 Hz, H-7a), 0.79 (1H, dt, J 12.6, 3.4, 3.4 Hz, H-
5endo). 13C NMR (125.73 MHz): 165.54 (COO), 133.38 (C-40),
130.49 (C-10), 129.27 (C-20), 128.93 (C-30), 72.92 (C-2), 68.86 (C-
6), 47.87 (C-1), 40.38 (C-3), 38.06 (C-5), 36.31 (C-4), 34.83 (C-7).
EI MS, m/z (rel%): 232 (37) [M], 188 (26), 166 (11), 123 (60), 110
(35), 105 (100) [Bz], 77 (60). For C14H16O3 (232.3) calcd: C,
72.39; H, 6.94; found: C, 72.12; H, 7.13.
8.79 (1H, s, H-20), 8.00–7.94 (2H, m, H-200), 7.68–7.64 (1H, m, H-
400), 7.56–7.50 (2H, m, H-300), 4.99–4.90 (1H, dm, J 7.1 Hz, H-2),
4.61 (1H, bdd, J 8.4, 3.9 Hz, H-5), 2.72 (1H, bd, J 5.0 Hz, H-4), 2.61
(1H, bd, J 5.0 Hz, H-1), 2.24 (1H, dt, J 14.2, 4.5, 4.5 Hz, H-6exo),
2.13–2.06 (2H, m, H-3endo, H-6endo), 1.79–1.68 (3H, m, H-3exo,
13
H-7). C NMR (125.73 MHz): 165.55 (COO), 152.26 (C-40), 151.50
(C-20), 149.19 (C-60), 145.70 (C-80), 133.53 (C-400), 131.53 (C-50),
130.13 (C-100), 129.35 (C-200), 128.95 (C-300), 76.42 (C-2), 57.27 (C-
5), 41.30 and 41.27 (C-1, C-4), 36.56 (C-3), 32.90 (C-7), 31.81 (C-
6). ESI MS, m/z (rel%): 369/371 (100/33) [M+H]. For C19H17ClN4O2
(368.8) calcd: C, 61.87; H, 4.65; Cl, 9.61; N, 15.19; found: C,
61.84; H, 4.63; Cl, 9.79; N, 15.14.
*
*
*
*
5.1.8. (1R ,2S ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
*
*
*
*
heptan-2-ol (31) and (1R ,2S ,4R ,5S )-5-(6-chloro-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (32)
*
*
*
*
5.1.6.2. (1R ,2S ,4R ,5R )-5-Hydroxybicyclo[2.2.1]hept-2-yl ben-
zoate (28). Yield 4.75 g (91%), colorless oil. 1H NMR
(499.95 MHz): 7.98–7.92 (1H, m, H-20), 7.67–7.63 (1H, m, H-40),
7.55–7.49 (2H, m, H-30), 4.81–4.75 (1H, dm, J 7.1 Hz, H-2), 4.69
(1H, d, J 4.1 Hz, OH), 4.03–5.97 (1H, m, H-5), 2.56 (1H, bd, J
5.7 Hz, H-1), 2.44 (1H, ddd, J 13.6, 7.1, 2.4 Hz, H-3endo), 2.22–
2.17 (1H, m, H-4), 1.89 (1H, ddd, J 13.2, 10.1, 5.5 Hz, H-6exo),
1.60–1.55 (1H, dm, J 10.2 Hz, H-7b), 1.38–1.30 (1H, dm, J 13.6 Hz,
H-3exo), 1.33–1.27 (1H, dm, J 10.1 Hz, H-7a), 0.71 (1H, dt, J 13.2,
3.5, 3.5 Hz, H-6endo). 13C NMR (125.73 MHz): 165.48 (COO),
133.44 (C-40), 130.36 (C-10), 129.28 (C-20), 128.95 (C-30), 77.54 (C-
2), 69.31 (C-5), 42.05 (C-1), 41.30 (C-4), 34.49 (C-6), 33.87 (C-7),
30.49 (C-3). EI MS, m/z (rel%): 232 (10) [M], 230 (8), 166 (15),
110 (90), 105 (100) [Bz], 77 (71). For C14H16O3 (232.3) calcd: C,
72.39; H, 6.94; found: C, 72.08; H, 7.03.
To a stirred solution of a benzoylated compound 29 or 30 (1.8 g,
4.88 mmol) in THF (55 mL) was added methylmagnesium chloride
(7.2 mL, 3 M solution in THF, 21.6 mmol) at 0 °C and the mixture
was stirred at 0 °C overnight. Reaction was quenched with diluted
AcOH (added until the inorganic precipitated salt dissolved again)
and reaction mixture was evaporated and adsorbed on silica gel
from minimum volume of water–methanol mixture. This silica
gel was placed on the top of the silica gel column (200 g). Chroma-
tography with ethyl acetate?ethyl acetate–toluene–acetone–eth-
anol (17:4:3:1) afforded product. Compounds were crystallized
from appropriate mixture of solvents.
*
*
*
*
5.1.8.1.
(1R ,2S ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo-
[2.2.1]heptan-2-ol (31). Yield 750 mg (58%). Mp 155–156 °C
(ethyl acetate–hexanes, white crystals). 1H NMR (499.95 MHz):
8.85 (1H, s, H-80), 8.77 (1H, s, H-20), 4.84 (1H, d, J 3.8 Hz, OH),
4.45 (1H, dd, J 8.4, 4.8 Hz, H-6), 3.89–3.86 (1H, m, H-2), 2.43–
2.37 (2H, m, H-1, H-4), 1.97–1.93 (1H, m, H-5exo), 1.87 (1H, ddd,
J 13.1, 8.5, 1.4 Hz, H-5endo), 1.68 (1H, ddd, J 12.9, 7.0, 1.6 Hz, H-
3endo), 1.67–1.63 (2H, m, H-7), 1.30–1.26 (1H, m, H-3exo). 13C
NMR (125.73 MHz): 152.13 (C-40), 151.45 (C-20), 149.15 (C-60),
145.69 (C-80), 131.45 (C-50), 71.00 (C-2), 54.67 (C-6), 50.25 (C-1),
40.51 (C-3), 37.00 (C-5), 34.96 (C-4), 32.42 (C-7). ESI MS, m/z
(rel%): 287/289 (40/32) [M+Na], 265/267 (100/30) [M+H]. For
C12H13ClN4O (264.7) calcd: C, 54.45; H, 4.95; Cl, 13.39; N, 21.17;
found: C, 54.28; H, 4.89; Cl, 13.73; N, 20.92.
*
*
*
*
5.1.7. (1R ,2S ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
*
*
*
*
hept-2-yl benzoate (29) and (1R ,2S ,4R ,5S )-5-(6-chloro-9H-
purin-9-yl)bicyclo[2.2.1]hept-2-yl benzoate (30)
To a mixture of the alcohol 27 or 28 (2.52 g, 10.8 mmol), tri-
phenylphosphine (3.7 g, 14.1 mmol), and 6-chloropurine (2.18 g,
14.1 mmol) in THF (50 mL) a solution of the diisopropyl azadicarb-
oxylate (2.8 mL, 14.2 mmol) in THF (30 mL) was dropwise added.
The resulting mixture was stirred overnight (TLC control) and then
heated to reflux for 3 hours. The reaction mixture was evaporated
and the residue was chromatographed on silica gel (200 g) in tolu-
ene–ethyl acetate (3:1).
*
*
*
*
5.1.8.2.
(1R ,2S ,4R ,5S )-5-(6-Chloro-9H-purin-9-yl)bicyclo-
*
*
*
*
5.1.7.1.
(1R ,2S ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo[-
[2.2.1]heptan-2-ol (32). Yield 930 mg (72%). Mp 202–203 °C
(ethyl acetate, white crystals). 1H NMR (600.13 MHz): 8.85 (1H, s,
H-80), 8.75 (1H, s, H-20), 4.73 (1H, d, J 3.7 Hz, OH), 4.42 (1H, ddd,
2.2.1]hept-2-yl benzoate (29). Yield 2.50 g (62.8%). Mp 162–
164 °C (water–ethanol (1:1), white crystals). 1H NMR

4382
M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
*
*
*
J 8.5, 3.9, 1.1 Hz, H-5), 3.73–3.69 (1H, m, H-2), 2.54 (1H, bd, J 5.0 Hz,
H-4), 2.22 (1H, bd, J 5.4 Hz, H-1), 2.02 (1H, dddd, J 13.9, 3.9, 5.0,
1.0 Hz, H-3exo), 1.83 (1H, ddd, J 13.9, 8.4, 2.2 Hz, H-6endo), 1.78
(1H, ddd, J 13.3, 6.8, 2.4 Hz, H-3endo), 1.68–1.62 (1H, dm, J
10.5 Hz, H-7b), 1.58–1.52 (1H, dm, J 10.4 Hz, H-7a), 1.31 (1H, dddd,
J 13.3, 5.0, 2.4, 1.0 Hz, H-3exo). 13C NMR (150.92 MHz): 152.22 (C-
40), 151.43 (C-20), 149.11 (C-60), 145.60 (C-80), 131.46 (C-50), 71.84
(C-2), 57.65 (C-5), 43.73 (C-1), 41.22 (C-4), 38.98 (C-3), 32.34 (C-
6), 31.84 (C-7). ESI MS, m/z (rel%): 288 (100), 265/267 (44/14)
[M+H]. For C12H13ClN4O (264.7) calcd: C, 54.45; H, 4.95; Cl,
13.39; N, 21.17; found: C, 54.75; H, 4.91; Cl, 13.19; N, 21.00.
5.1.10.1.
6-Chloro-9-[(1R ,2R ,4S )-6,6-difluorobicyclo[2.2.1]
hept-2-yl]-9H-purine (35). Yield 152 mg (55%). Mobile phase:
toluene–ethyl acetate (7:1?4:1). Mp 156–158 °C (water–metha-
nol, white solid). 1H NMR (499.95 MHz): 8.93 (1H, s, H-8), 8.80
(1H, s, H-2), 5.06–5.00 (1H, m, H-20), 2.96 (1H, d, J(10,F) 8.0 Hz, H-
10), 2.66–2.60 (1H, m, H-40), 2.32–2.22 (2H, m, H-30), 2.08 m, 1H
(H-50exo), 2.03–1.98 (1H, m, H-70b), 1.89–1.83 (1H, m, H-50endo),
1.78–1.70 (1H, dm, J 11.5 Hz, H-70a). 13C NMR (125.73 MHz):
152.25 (C-4), 151.66 (C-2), 149.21 (C-6), 145.87 (C-8), 131.46 (C-
5), 130.38 (dd, J(60,F) 256.4 and 252.9 Hz, C-60), 51.42 (t, J(20,F)
8.0 Hz, C-20), 50.69 (dd, J(10,F) 19.4 and 25.6 Hz, C-10), 41.35 (dd,
J(50,F) 21.9 and 23.5 Hz, C-50), 35.83 (m, C-30, C-40), 34.80 (d,
J(70,F) 4.0 Hz, C-70). ESI MS, m/z (rel%): 285/287 (100/33) [M+H].
For C12H11ClF2N4 (284.7) calcd: C, 50.63; H, 3.89; Cl, 12.45; F,
13.35; N, 19.68; found: C, 50.87; H, 3.85; N, 19.51.
*
*
*
5.1.9. (1R ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
*
*
*
heptan-2-one (33) and (1R ,4R ,5S )-5-(6-chloro-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-one (34)
A mixture of PDC (3.28 g, 8.6 mmol), molecular sieves (3A, pow-
der, 5 g) and dichloromethane (50 mL) was stirred at room temper-
ature for 15 min. A solution of alcohol 31 or 32 (1.15 g, 4.34 mmol)
in a mixture of dichloromethane (50 mL) and chloroform (20 mL)
was added to the reaction mixture. After 5 days of stirring at room
temperature, the mixture was filtered and the filtrates were evap-
orated. The residue was chromatographed on silica gel (200 g).
* * *
6-Chloro-9-[(1R ,2S ,4R )-5,5-difluorobicyclo[2.2.1]
5.1.10.2.
hept- 2-yl]-9H-purine (36). Yield 124 mg (45%). Mobile phase:
toluene–ethyl acetate (7:1?4:1). Mp 163–164 °C (water–metha-
nol, white crystals). 1H NMR (499.95 MHz): 8.90 (1H, s, H-8),
8.79 (1H, s, H-2), 4.78 (1H, ddd, J 8.2, 4.5, 1.2 Hz, H-20), 2.81 (1H,
bd, J 4.6 Hz, H-10), 2.71–2.65 (1H, m, H-40), 2.37–2.13 (3H, m, H-
30exo, 30endo, H-60exo), 2.02–1.92 (2H, m, H-60endo, H-70b), 1.77–
1.69 (1H, dm, J 11.1 Hz, H-70a). 13C NMR (125.73 MHz): 152.22
(C-4),151.54 (C-2), 149.23 (C-6), 145.83 (C-8), 131.55 (C-5),
130.76 (dd, J(50,F) 254.4 and 251.6 Hz, C-50), 56.51 (d, J(20,F)
1.4 Hz (C-20), 44.40 (dd, J(40,F) 24.8 and 21.0 (C-40), 41.72 (t,
J(10,F) 3.2 Hz, C-10), 40.45 (t, J(60,F) 24.1 Hz, C-60), 34.34 (d, J(70,F)
4.9 Hz, C-70), 28.89 (dd, J(30,F) 6.8 and 5.3 Hz, C-30). ESI MS, m/z
(rel%): 285/287 (100/35) [M+H]. For C12H11ClF2N4 (284.7) calcd:
C, 50.63; H, 3.89; Cl, 12.45; F, 13.35; N, 19.68; found: C, 50.67; H,
3.99; N, 19.31.
*
*
*
5.1.9.1. (1R ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
heptan-2-one (33). Yield 1.01 g (88%). Mobile phase: toluene–
ethyl acetate (3:2). Mp 181.5–182.5 °C (ethyl acetate, white pow-
1
der). H NMR (499.95 MHz): 8.94 (1H, s, H-80), 8.79 (1H, s, H-20),
4.88 (1H, bdd, J 8.5, 4.4 Hz, H-6), 2.92 (1H, bs, H-1), 2.90–2.86
(1H, m, H-4), 2.46–2.42 (1H, m, H-5exo), 2.33 (1H, ddd, J 13.6,
8.5, 2.2 Hz, H-5endo), 2.21–2.16 (2H, m, H-3exo, H-7b), 2.00 (1H,
dd, J 17.9, 4.4 Hz, H-3endo), 1.91–1.83 (1H, dm, J 11.1 Hz, H-7a).
13C NMR (125.73 MHz): 214.26 (C-2), 152.16 (C-40), 151.64 (C-20),
149.24 (C-60), 145.86 (C-80), 131.54 (C-50), 56.36 (C-1), 53.27 (C-
6), 43.84 (C-3), 35.67 (C-5), 35.41 (C-4), 35.40 (C-7). ESI MS, m/z
(rel%): 263/265 (100/34) [M+H]. For C12H11ClN4O (262.7) calcd:
C, 54.87; H, 4.22; Cl, 13.50; N, 21.33; found: C, 54.55; H, 4.03; Cl,
13.90; N, 20.94.
*
*
*
*
5.1.11. (1R ,2R ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo
*
*
*
*
[2.2.1]heptan-2-ol (37) and (1R ,2R ,4R ,5S )-5-(6-chloro-9H-
purin-9-yl)bicyclo[2.2.1]heptan-2-ol (38)
Ketoderivative 33 or 34 (440 mg, 1.67 mmol) was dissolved in
methanol (40 mL) and the solution was cooled to 0 °C, when so-
dium borohydride (43 mg, 1.14 mmol) was added. The reaction
mixture was stirred at 0 °C for 1 hour and evaporated, co-distilled
with methanol (50 mL) and evaporated. The residue was chro-
matographed on silica gel (120 g) in ethyl acetate?ethyl ace-
tate–toluene–acetone–ethanol (17:4:3:1) to afford endo-alcohol.
A small amount of the exo-alcohols 31 (17%) or 32 (20%) was ob-
tained from the reaction mixture as well.
*
*
*
5.1.9.2. (1R ,4R ,5S )-5-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]
heptan-2-one (34). Yield 1.05 g (92%). Mobile phase: toluene–
ethyl acetate (4:1). Mp 153–154 °C (ethyl acetate, white powder).
1H NMR (499.95 MHz): 8.95 (1H, s, H-80), 8.80 (1H, s, H-20), 4.89
(1H, ddd, J 8.3, 4.3, 1.2, H-5), 3.02 (1H, bd, J 5.4 Hz, H-4), 2.66
(1H, bd, J 4.9 Hz, H-1), 2.48–2.44 (1H, m, H-6exo), 2.29–2.25 (1H,
m, H-3exo), 2.22 (1H, ddd, J 14.1, 8.4, 2.3 Hz, H-6endo), 2.13–2.07
(2H, m, H-3endo, H-7b), 1.89–1.81 (1H, dm, J 12.1 Hz, H-7a). 13C
NMR (125.73 MHz): 214.85 (C-2), 152.28 (C-40), 151.57 (C-20),
149.23 (C-60), 145.86 (C-80), 131.55 (C-50), 56.57 (C-5), 49.29 (C-
1), 42.19 (C-3), 41.31 (C-4), 34.70 (C-7), 31.20 (C-6). ESI MS, m/z
(rel%): 263/265 (100/32) [M+H], 437 (82). For C12H11ClN4O
(262.7) calcd: C, 54.87; H, 4.22; Cl, 13.50; N, 21.33; found: C,
54.95; H, 4.23; Cl, 13.50; N, 21.00.
*
*
*
*
5.1.11.1. (1R ,2R ,4S ,6R )-6-(6-Chloro-9H-purin-9-yl)bicyclo-
[2.2.1]heptan-2-ol (37). Yield 293 mg (66%). Mp 156–157 °C
(ether–hexanes, white crystals). 1H NMR (499.84 MHz): 8.86 (1H,
s, H-80), 8.78 (1H, s, H-20), 5.30 (1H, bdd, J 8.8, 4.5 Hz, H-6), 5.10
(1H, d, J 3.8 Hz, OH), 4.21–4.16 (1H, m, H-2), 2.57–2.51 (1H, m,
H-1), 2.36–2.32 (1H, m, H-4), 2.12 (1H, ddd, J 12.8, 8.7, 2.3 Hz, H-
5endo), 1.99–1.92 (1H, m, H-5exo), 1.94–1.88 (1H, m, H-3endo),
1.89–1.81 (1H, dm, J 10.7 Hz, H-7b), 1.46–1.37 (1H, dm, J 10.7 Hz,
*
*
*
5.1.10. 6-Chloro-9-[(1R ,2R ,4S )-6,6-difluorobicyclo[2.2.1]
hept-2-yl]-9H-purine (35) and 6-chloro-9-[(1R ,2S ,4R )-5,5-
difluorobicyclo[2.2.1]hept-2-yl]-9H-purine (36)
H-7a), 0.88 (1H, dt, J
12.6, 3.5, 3.5 Hz, H-3exo). 13C NMR
*
*
*
(125.70 MHz): 152.17 (C-40), 151.50 (C-20), 149.13 (C-60), 146.04
(C-80), 131.29 (C-50), 69.49 (C-2), 50.94 (C-6), 48.50 (C-1), 39.57
(C-5), 37.59 (C-3), 36.99 (C-4), 35.28 (C-7). ESI MS, m/z (rel%):
265/267 (100/33) [M+H]. For C12H13ClN4O (264.7) calcd: C,
54.45; H, 4.95; Cl, 13.39; N, 21.17; found: C, 54.65; H, 5.15; Cl,
13.67; N, 20.87.
To a stirred mixture of the ketone 33 or 34 (255 mg, 0.97 mmol)
in dichloromethane (10 mL) and pyridine (1.2 mL), DAST (1.9 mL,
14.4 mmol) was added at room temperature under argon. The
reaction mixture was refluxed under argon for 30 hours. After cool-
ing to room temperature, the reaction mixture was poured onto a
saturated solution of NaHCO3 + ice (50 mL). The water phase was
extracted with ethyl acetate (100 mL). The organic phase was
washed with saturated NaHCO3 (50 mL) and water (50 mL), dried
over anhydrous sodium sulfate and evaporated. Residue was chro-
matographed on silica gel (150 g).
*
*
*
*
5.1.11.2. (1R ,2R ,4R ,5S )-5-(6-Chloro-9H-purin-9-yl)bicyclo-
[2.2.1]heptan-2-ol (38). Yield 280 mg (63%). Mp 172.5–174 °C
(ethyl acetate, white crystals). 1H NMR (600.13 MHz): 8.87 (1H, s,
H-80), 8.77 (1H, s, H-20), 4.83 (1H, d, J 4.0 Hz, OH), 4.58 (1H, ddd,

M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
4383
J 8.6, 4.2, 1.2 Hz, H-5), 4.12–4.06 (1H, m, H-2), 2.63 (1H, ddd, J 13.4,
8.6, 2.4 Hz, H-6endo), 2.44 (1H, bd, J 5.1 Hz, H-4), 2.35–2.30 (1H, m,
H-1), 1.98 (1H, ddd, J 13.1, 10.1, 5.2 Hz, H-3exo), 1.91 (1H, dtt, J
13.5, 4.2, 4.2, 1.1, 1.1 Hz, H-6exo), 1.75–1.67 (1H, dm, J 10.7 Hz,
H-7b), 1.41–1.33 (1H, dm, J 10.7 Hz, H-7a), 0.95 (1H, dt, J 13.1,
3.5, 3.5 Hz, H-3endo). 13C NMR (150.92 MHz): 152.15 (C-40),
151.49 (C-20), 149.14 (C-60), 145.70 (C-80), 131.45 (C-50), 69.39 (C-
2), 58.28 (C-5), 42.92 (C-4), 42.14 (C-1), 37.44 (C-3), 34.58 (C-7),
28.93 (C-6). ESI MS, m/z (rel%): 265/267 (100/33) [M+H]. For
C12H13ClN4O (264.7) calcd: C, 54.45; H, 4.95; Cl, 13.39; N, 21.17;
found: C, 54.41; H, 4.78; Cl, 13.20; N, 21.05.
1.66 mmol) in THF (30 mL). The resulting mixture was stirred over-
night and then heated to reflux for 10 hours. The reaction mixture
was evaporated and the residue was chromatographed on silica gel
column (200 g) in ethyl acetate?ethyl acetate–toluene–acetone–
ethanol (17:4:3:1). 303 mg (50%) of the product 41 was obtained.
Compound was crystallized from water–methanol. Mp 272–
274 °C (decomposition, white powder). 1H NMR (499.84 MHz):
8.89 (2H, s, H-8), 8.80 (2H, s, H-2), 4.93 (2H, dd, J 8.0, 4.5 Hz, H-
20, H-60), 3.14 (1H, bs, H-10), 2.66 (1H, m, H-40), 2.24–2.14 (4H, m,
H-30, H-50), 1.92 (2H, bs, H-70). 13C NMR (125.70 MHz): 152.17
(C-4), 151.62 (C-2), 149.26 (C-6), 145.67 (C-8), 131.48 (C-5),
56.29 (C-20 and C-60), 47.72 (C-10), 36.85 (C-30 and C-50), 36.01
*
*
*
*
5.1.12. 6-Chloro-9-[(1S ,2S ,4R ,6R )-6-fluorobicyclo[2.2.1]
(C-40), 34.12 (C-70). ESI MS, m/z (rel%): 401/403 (100/60) [M+H].
*
*
*
*
3
hept-2-yl]-9H-purine (39) and 6-chloro-9-[(1R ,2S ,4R ,5S )-5-
fluorobicyclo[2.2.1]hept-2-yl]-9H-purine (40)
For C17H14Cl2N8 ꢀ
H2O (414.8) calcd: C, 48.01; H, 3.95; Cl,
4
16.67; N, 26.35; found: C, 48.01; H, 3.95; Cl, 16.95; N, 26.15.
To
a stirred mixture of endo-alcohol 37 or 38 (300 mg,
*
*
*
*
1.13 mmol) in dichloromethane (10 mL) and pyridine (1.2 mL),
DAST (0.7 mL, 5.3 mmol) was added at room temperature under
argon. The reaction mixture was refluxed under argon for 12 hours.
After cooling to room temperature., the reaction mixture was
poured onto saturated solution of NaHCO3 + ice (50 mL).The water
phase was extracted with ethyl acetate (100 mL). The organic
phase was washed with saturated NaHCO3 (50 mL) and water
(50 mL), dried over anhydrous sodium sulfate and evaporated. Res-
idue was chromatographed on silica gel (150 g) in toluene–ethyl
acetate (2:1) to afford product.
5.1.14. 9,90-(1R ,2S ,4R ,5S )-Bicyclo[2.2.1]heptane-2,5-diylbis-
(6-chloro-9H-purine) (42)
A
solution of the diisopropyl azadicarboxylate (0.4 mL,
1.96 mmol) in THF (10 mL)was dropwise added to a mixture of
the hydroxyderivative 38 (400 mg, 1.51 mmol), triphenylphos-
phine (515 mg, 1.96 mmol) and 6-chloropurine (267 mg, 1.66
mmol) in THF (30 mL). The resulting mixture was stirred overnight
and then heated to reflux for 10 hours. The reaction mixture was
evaporated to the one half and the solid product was filtered off,
washed with THF, methanol and water. 150 mg (25%) of the prod-
uct 42 was obtained. The filtrates were evaporated and the residue
was chromatographed on silica gel column (200 g) in ethyl ace-
tate?ethyl acetate–toluene–acetone–ethanol (17:4:3:1). 180 mg
(30%) of second crop of the product 42 was obtained. Mp >300 °C
(THF, white powder). 1H NMR (499.95 MHz): 8.92 (2H, s, H-8),
8.81 (2H, s, H-2), 4.78 (2H, dd, J 7.3, 5.6 Hz, H-20, H-50), 2.87–2.83
(2H, m, H-10, H-40), 2.32–2.30 (4H, m, H-30, H-60), 1.89 (2H, bs, H-
70). 13C NMR (125.73 MHz): 152.27 (C-4), 151.52 (C-2), 149.20
(C-6), 145.77 (C-8), 131.54 (C-5), 57.33 (C-20 and C-50), 42.14 (C-
10 and C-40), 34.76 (C-30 and C-60), 33.66 (C-70). ESI MS, m/z
(rel%): 401/403 (94/56) [M+H]. For C17H14Cl2N8 (401.3) calcd: C,
50.89; H, 3.52; Cl, 17.67; N, 27.93; found: C, 50.86; H, 3.38; Cl,
17.99; N, 27.62.
*
*
*
*
5.1.12.1.
6-Chloro-9-[(1S ,2S ,4R ,6R )-6-fluorobicyclo[2.2.1]
hept-2-yl]-9H-purine (39). Yield 219 mg (73%). Mp 127.5–
129 °C (water–methanol, white crystals). 1H NMR (499.95 MHz):
8.87 (1H, s, H-8), 8.79 (1H, s, H-2), 4.98 (1H, ddm, J(60,F) 54.5,
6.4 Hz, H-60), 4.52 (1H, dd, J 8.3, 5.0 Hz, H-20), 2.85 (1H, bd, J(10,F)
8.8 Hz, H-10), 2.54–2.49 (1H, m, H-40), 2.02–1.98 (1H, m, H-30exo),
1.93 (1H, ddd, J 13.0, 8.3, 2.2 Hz, H-30endo), 1.88–1.84 (1H, m, H-
50endo), 1.87–1.81 (1H, m, H-70b), 1.64–1.59 (1H, m, H-70a), 1.63–
1.57 (1H, m, H-50exo). 13C NMR (125.73 MHz): 152.15 (C-4),
151.51 (C-2), 149.19 (C-6), 145.79 (C-8), 131.49 (C-5), 93.71 (d,
J(60,F) 181.6 Hz, C-60), 52.85 (d, J(20,F) 14.5 Hz, C-20), 48.11 (d,
J(10,F) 21.9 Hz, C-10), 38.45 (d, J(50,F) 19.7 Hz, C-50), 36.54 (C-30),
34.74 (C-40), 32.90 (C-70). ESI MS, m/z (rel%): 267/269 (76/23)
[M+H], 288 (100). For C12H12ClFN4 (266.7) calcd: C, 54.04; H,
4.54; Cl, 13.29; F, 7.12; N, 21.01; found: C, 53.92; H, 4.31; N, 20.85.
*
*
* *
5.1.15. 9,90-(1R ,2S ,3S ,4S)-Bicyclo[2.2.1]hept-5-ene-2,3-diylbis-
(6-chloro-9H-purine) (45)
*
*
*
*
A mixture of (1R ,2S ,3S ,4S )-bicyclo[2.2.1]hept-5-ene-2,3-dia-
mine13 43 (750 mg, 6 mmol), 4,6-dichloropyrimidin-5-amine
(3.94 g, 24 mmol), and triethylamine (3.6 mL) in ethanol (9 mL)
was heated in a pressure vessel at 105 °C for 10 days and, after
cooling, was evaporated. The residue was chromatographed on a
column of silica gel (200 g). The pyrimidine intermediate was
eluted with toluene–ethyl acetate (4:1) and this intermediate
was immediately used in the next step. Concentrated hydrochloric
acid (2 mL) was added to a suspension of pyrimidine intermediate
in triethyl orthoformate (160 mL) and THF (30 mL) and the reac-
tion mixture was vigorously stirred for 3 days at room tempera-
ture. Reaction mixture was evaporated and residue was
chromatographed on silica gel column in ethyl acetate to afford
450 mg (19%) of the product 45. Mp 219–220.5 °C (water–metha-
nol, white powder). 1H NMR (499.84 MHz): 9.01 (1H, s, H-8),
8.80 (1H, s, H-800), 8.63 (1H, s, H-200), 8.55 (1H, s, H-2), 6.70 (1H,
dd, J 5.6, 3.3 Hz, H-60), 6.30 (1H, dd, J 5.7, 2.8 Hz, H-50), 5.75 (1H,
t, J 3.7, 3.7 Hz, H-30), 5.15 (1H, dd, J 3.8, 2.2 Hz, H-20), 3.65–3.62
(1H, m, H-10), 3.57–3.55 (1H, m, H-40), 2.51–2.47 (1H, m, H-70b),
1.92–1.86 (1H, dm, J 9.9 Hz, H-70a). 13C NMR (125.70 MHz):
152.68 and 152.63 (C-4 and C-400), 151.58 and 151.48 (C-2 and C-
200), 149.22 (C-6 and C-600), 146.19 (C-8), 145.68 (C-800), 138.28 (C-
60), 136.05 (C-50), 131.55 (C-5), 131.09 (C-500), 61.15 (C-20), 60.91
(C-30), 47.86 (C-10), 46.72 (C-40), 46.25 (C-70). ESI MS, m/z (rel%):
*
*
*
*
5.1.12.2.
6-Chloro-9-[(1R ,2S ,4R ,5S )-5-fluorobicyclo[2.2.1]
hept-2-yl]-9H-purine (40). Yield 190 mg (63%). Mp 111–112 °C
(water–methanol, white crystals). 1H NMR (499.95 MHz): 8.87
(1H, s, H-8), 8.77 (1H, s, H-2), 4.81 (1H, ddm, J(50,F) 56.0, 6.3 Hz,
H-50), 4.47 (1H, ddm, J 8.5, 3.8 Hz, H-20), 2.69 (1H, bd, J 5.0 Hz, H-
10), 2.61–2.56 (1H, m, H-40), 2.18–2.13 (1H m, H-30exo), 1.99 (1H,
ddd, J(60en,F) 18.2, 14.4, 6.3, 2.7 Hz, H-60endo), 1.88 (1H, ddm, J
14.4, 8.3 Hz, H-30endo), 1.76–1.68 (1H, dm, J 11.0 Hz, H-70b), 1.64
(1H, ddt, J(60ex,F) 39.1, 14.4, 5.1, 1.5, 1.5 (H-60exo), 1.63–1.57 (1H,
m, H-70a). 13C NMR (125.73 MHz): 152.26 (C-4), 151.46 (C-2),
149.17 (C-6), 145.68 (C-8), 131.50 (C-5), 94.46 (d, J(50,F) 180.8 Hz,
C-50), 57.03 (C-20), 41.74 (d, J(40,F) 20.7 Hz, C-40), 40.75 (C-10),
36.94 (d, J(60,F) 20.7 Hz, C-60), 32.22 (C-70), 29.96 (d, J(30,F)
11.5 Hz, C-30). ESI MS, m/z (rel%): 267/269 (100/33) [M+H]. For
C12H12ClFN4 (266.7) calcd: C, 54.04; H, 4.54; Cl, 13.29; F, 7.12; N,
21.01; found: C, 53.91; H, 4.31; N, 20.71.
*
*
5.1.13. 9,90-(2R ,6S)-Bicyclo[2.2.1]heptane-2,6-diylbis(6-chloro-
9H-purine) (41)
A
solution of the diisopropyl azadicarboxylate (0.4 mL,
1.96 mmol) in THF (10 mL) was added dropwise to a mixture of
the hydroxyderivative 37 (400 mg, 1.51 mmol), triphenylphos-
phine (515 mg, 1.96 mmol) and 6-chloropurine (267 mg,

4384
M. Šála et al. / Bioorg. Med. Chem. 18 (2010) 4374–4384
399/401 (100/63) [M+H]. For C17H12Cl2N8 (399.2) calcd: C, 51.14;
H, 3.03; Cl, 17.76; N, 28.07; found: C, 51.02; H, 2.86; Cl, 17.81; N,
27.85.
5.2.2. Cytostatic activity assays
The cytotoxicity of the compounds was evaluated by the MTS-
method and the 50% cytotoxic concentration (CC50) was calculated.
Briefly, the same experimental set-up was used as for the antiviral
assay, but for cytotoxicity determination, uninfected cultures were
incubated with serial dilution of compound for three days at 37 °C.
The cytotoxic activity was calculated using the following formula:
*
*
*
*
5.1.16. 9,90-(1R ,2S ,3S ,4S )-Bicyclo[2.2.1]heptane-2,3-diyl-
bis(6-chloro-9H-purine) (46)
*
*
*
*
A
mixture of (1R ,2S ,3S ,4S )-bicyclo[2.2.1]heptane-2,3-dia-
*
mine14 44 (750 mg, 6 mmol), 4,6-dichloropyrimidin-5-amine
(3.94 g, 24 mmol), and triethylamine (3.6 mL) in ethanol (9 mL)
was heated in a pressure vessel at 105 °C for 10 days and, after
cooling, was evaporated. The residue was chromatographed on a
column of silica gel (200 g). Pyrimidine intermediate was eluted
with toluene–ethyl acetate (4:1) and this intermediate was
immediately used in the next step. Concentrated hydrochloric
acid (2 mL) was added to a suspension of pyrimidine intermedi-
ate in triethyl orthoformate (160 mL) and THF (30 mL) and the
reaction mixture was vigorously stirred for 3 days at room tem-
perature. The reaction mixture was evaporated and residue was
chromatographed on silica gel column in ethyl acetate to afford
490 mg (20%) of the product 46. Mp 191–192.5 °C (water–meth-
anol, white powder). 1H NMR (499.95 MHz): 9.03 (1H, s, H-8),
8.95 (1H, s, H-800), 8.69 (1H, s, H-2), 8.56 (1H, s, H-200), 5.53
(1H, dd, J 5.0, 1.9 Hz, H-30), 5.45–5.41 (1H, m, H-20), 3.00–2.96
(2H, m, H-10, H-40), 2.45–2.39 (1H, dm, J 10.9 Hz, H-70b), 1.97–
1.93 (1H, m, H-50endo), 1.83–1.79 (1H, m, H-50exo), 1.69–1.63
(1H, dm, J 10.9 Hz, H-70a), 1.56–1.52 (1H, m, H-60exo), 1.28–
1.24 (1H, m, H-60endo). 13C NMR (125.73 MHz): 152.75 (C-4),
152.25 (C-400), 151.51 and 151.42 (C-2 and C-200), 149.31 (C-6),
149.14 (C-600), 146.92 (C-8), 145.81 (C-800), 131.42 and 131.36
(C-5 and C-500), 64.94 (C-20), 60.19 (C-30), 42.25 and 42.02 (C-10
and C-40), 35.97 (C-70), 27.33 (C-50), 21.28 (C-60). ESI MS, m/z
(rel%): 401/403 (100/65) [M+H]. For C17H14Cl2N8 (401.3) calcd:
C, 50.89; H, 3.52; Cl, 17.67; N, 27.93; found: C, 50.68; H, 3.50;
Cl, 17.87; N, 27.59.
% CPE = 100 [ODCC ꢁ ODCompound]/ODCC, where ODCC corresponds
to the optical density of the uninfected and untreated cell cultures
and ODCompound are uninfected cultures, treated with compound.
Acknowledgments
This work is a part of the research project Z4 055 0506. It was
supported by the ‘Centre for New Antivirals and Antineoplastics’
(Ministry of Education, Youth and Sports of the Czech Republic,
1M0508) and by Gilead Sciences, Inc. (Foster City, CA, U.S.A.).
Armando M. De Palma is a postdoctoral fellow of the FWO-Vlaand-
eren. The author (M.S.) would like to thank to Ms. Eliška Procház-
ková for her crucial role in preparing of the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.081.
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90 lL medium and 10 lL MTS/PMS (Promega, Leiden, The Nether-
lands) were added to each well. After an incubation period of 2
hours at 37 °C the optical density of each well was read at
498 nm in a microplate reader. CPE values were calculated as fol-
*
lows:
%
CPE = 100 [ODCC ꢁ ODCVB3+Compound]/[ODCC ꢁ ODVC]. In
these formulae, ODCC corresponds to the optical density of the
uninfected and untreated cell cultures, ODVC represents the in-
fected and untreated cell cultures and ODCVB3+Compound are CVB3-
infected cell cultures, treated with a given concentration of
compound.