From norbornane-based nucleotide analogs locked in South conformation to novel inhibitors of feline herpes virus

Article abstract of DOI:10.1016/j.bmc.2014.04.004

A synthetic route toward a series of unique cyclic nucleoside phosphonates locked in South conformation is described. The desired conformation is stabilized by a substitution of the sugar moiety by bicyclo[2.2.1]heptane (norbornane) bearing a purine or pyrimidine nucleobase in the bridgehead position. Although the final phosphonate derivatives are devoid of any significant antiviral activity probably due to the unfavorable conformational properties, several intermediates and their analogs exhibit surprising activity against feline herpes virus. Since these compounds do not possess an appropriate hydroxymethyl function allowing phosphorylation and subsequent incorporation into the polynucleotide chain, it seems to be likely that these compounds act by a novel unknown mechanism of action and may represent a new possible alternative for nucleoside and nucleotide therapeutics of this widely spread feline infection. A number of derivatives exerted also a significant antiviral activity against Coxsackievirus B3 and B4.

Full text of DOI:10.1016/j.bmc.2014.04.004

Bioorganic & Medicinal Chemistry 22 (2014) 2974–2983
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
From norbornane-based nucleotide analogs locked in South
conformation to novel inhibitors of feline herpes virus
a
a
a
a
a
ˇ
´
ˇ
´
Milan Dejmek , Hubert Hrebabecky , Michal Šála , Martin Dracínsky , Eliška Procházková ,
Pieter Leyssen b, Johan Neyts b, Jan Balzarini b, Radim Nencka a,
⇑
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, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic route toward a series of unique cyclic nucleoside phosphonates locked in South conformation
is described. The desired conformation is stabilized by a substitution of the sugar moiety by bicy-
clo[2.2.1]heptane (norbornane) bearing a purine or pyrimidine nucleobase in the bridgehead position.
Although the final phosphonate derivatives are devoid of any significant antiviral activity probably due
to the unfavorable conformational properties, several intermediates and their analogs exhibit surprising
activity against feline herpes virus. Since these compounds do not possess an appropriate hydroxymethyl
function allowing phosphorylation and subsequent incorporation into the polynucleotide chain, it seems
to be likely that these compounds act by a novel unknown mechanism of action and may represent a new
possible alternative for nucleoside and nucleotide therapeutics of this widely spread feline infection. A
number of derivatives exerted also a significant antiviral activity against Coxsackievirus B3 and B4.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 17 January 2014
Revised 31 March 2014
Accepted 3 April 2014
Available online 13 April 2014
Keywords:
Carbocyclic nucleosides
Nucleoside phosphonates
Purine
Norbornane
Feline herpes virus
1. Introduction
NH2
N
HO
HO
NH2
N
N
N
N
O
P
N
N
Modern antiviral treatment relies on nucleoside and nucleotide
derivatives, a fundamental element of so-called ‘cocktail therapy’,
which is the most successful in the current therapy of HIV infec-
tion. The development of anti-HIV therapeutics has brought nucle-
oside phosphonates to the spotlight and allowed their full
recognition as the most effective class of nucleoside and nucleotide
reverse-transcriptase inhibitors (NRTIs).1 Although the acyclic
nucleoside phosphonates (ANPs), represented by tenofovir 1, have
obtained most of the fame, there is a number of other interesting
cyclic nucleoside phosphonates being developed including GS-
9148 (2) and its carbocyclic derivatives (e.g., 3), which combine
the strengths of the nucleoside phosphonates, the didanosine or
abacavir-like skeleton and rational design-based optimization,
leading to their significant anti-HIV potency and favorable resis-
tance profile.2 Another cyclic nucleoside phosphonate that has
gained considerable attention in the literature is PMDTA (4),
reported by Herdewijn and co-workers (Fig. 1).3
HO
HO
O
N
O
P
X
O
F
1
2
3
X=O
X=CH2
NH2
N
N
HO
HO
N
N
O
P
O
O
4
Figure 1. The structures of important acyclic and cyclic nucleoside phosphonates.
Saneyoshi et al. have prepared several locked derivatives of
PMDTA,4 whereas Gilead Sciences, Inc. has filed a patent concern-
ing various conformationally locked phosphonate analogs as anti-
viral agents.5
In both of these cases, however, the conformation was locked
using bicycle[3.1.0]hexane as a substitute of the natural tetrahy-
dofuran sugar ring. Recently, we have introduced bicy-
Although conformational constriction has proven to be a vital
modification of nucleosides leading to an enhancement of desired
antiviral properties, conformationally locked derivatives of cyclic
nucleoside phosphonate have been reported rather rarely so far.
clo[2.2.1]heptane (norbornane)6 as
a
potentially attractive
surrogate of the sugar moiety and shown that some derivatives
bearing the 6-chloropurine nucleobase can possess significant anti-
viral activities against (+)ssRNA viruses from the Picornaviridae
⇑
Corresponding author. Tel.: +420 220 183 265; fax: +420 220 183 560.
E-mail address: nencka@uochb.cas.cz (R. Nencka).
http://dx.doi.org/10.1016/j.bmc.2014.04.004
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
2975
family.7 This pseudosugar motif was adopted also by other authors
recently.8
Herein, we examine the use of norbornane skeleton for the sta-
9
14
a
a
X
N
X
N
N
N
bilization of the South conformation of racemic 50-nornucleosides
and the corresponding nucleoside phosphonates. Although the
findings of Marquez and co-workers suggest that the nucleosides
locked in South conformation are not suitable substrates for poly-
merases, which results in their inactivity against herpes viruses,9
we decided to investigate this trend further by employing a struc-
turally different type of bridges locking nucleoside phosphonates
in this particular conformation. To our surprise, several precursors
of this synthesis exhibit significant activity against feline herpes
virus (FHV-1), which is the most widely spread infection of house-
hold cats, causing typically severe respiratory and ophthalmologi-
cal problems in newborn kittens and resulting in a mortality of up
to 50%.10 The treatment of this disease has been traditionally con-
nected with nucleoside derivatives (trifluridine, ganciclovir)
N
N
N
N
O
NH2
O
18 X=Cl
19 X=NH2
b
c
d
e
23 X=Cl
24 X=OH
g
20 X=NHcPr
21
22
X=NMe2
X=SH
f
f
X
N
X
N
N
N
N
N
N
N
NH2
HO
HO
25 X=Cl
b
c
d
e
30
31X=OH
X=Cl
-lysine.11
g
26 X=NH2
27 X=NHcPr
28 X=NMe2
29 X=SH
together with adjuvant therapy by interferons and
L
2. Results and discussion
2.1. Chemistry
Scheme 2. Reagents and conditions: (a) PDC, DCM, 7 d, 82% for 18, or PDC, DMF,
12 h, 56% for 23; (b) NH3, EtOH, 120 °C, 30 min, 77% for 19, 70% for 26; (c)
cyclopropylamine, EtOH, MW, 140 °C, 30 min, 77% for 20, 85% for 27; (d) DMF, MW,
200 °C, 2 min, 86% for 21, 77% for 28; (e) thiourea, EtOH, 105 °C, overnight, 89% for
22, 68% for 29; (f) NaBH4, MeOH, 5 h, 89% for 25, 51% for 30; (g) TFA, H2O, 24 h, 86%
for 24, 64% for 31.
Using a methodology reported elsewhere, we were able to
obtain large quantities of norbornane intermediate 5,12 which
was selected as a suitable starting material for the further synthe-
sis of derivatives locked in South conformation. The preparation of
the crucial intermediate 6 was achieved using a six-step procedure
described in detail in Supplementary material. The essential step,
the introduction of a carboxylic moiety into the bridgehead posi-
tion, was performed by the Hell–Volhard–Zelinsky bromination
connected with the skeleton rearrangement.13
Since the target structures bear the nucleobase in the bridge-
head position of the norbornane bicycle, the only way to introduce
purine or thymine functionality is the nucleobase construction on a
primary amino group. In the case of 6-chloropurine, the classical
two-step procedure using triethyl orthoformate to close the imid-
azole ring was used; in the case of 2-amino-6-chloropurine, how-
ever, we advantageously employed a recently published one-pot
methodology, which uses a formylated pyrimidine precursor.14
All constructions of nucleobases proceeded smoothly with moder-
ate yields, which were expected with a highly sterically demanding
substrate such as 7 with the amino group on the tertiary carbon.
The amino group-containing key intermediate 7 was prepared
from 6 using Curtius rearrangement, which afforded 7 in good
yield (Scheme 1).
To explore the relationship between the structure and
biological activity further, and in order to enrich our library of
compounds, additional derivatization of
9
and 14 was
performed—first, the configuration of the C-2 hydroxy group was
inverted using a standard oxidation–reduction sequence employ-
ing PDC as the oxidant and NaBH4 as the reductant (Scheme 2).
Subsequently, the nucleophilic substitution of the C-6 chlorine
atom of the purine nucleobase of 9, 14, 18, 23, 25 and 30 gave rise
to a small library of final compounds. It is noteworthy that
although the reduction of the C-2 keto group of 18 with sodium
borohydride afforded the endo derivative almost exclusively (<3%
of the exo derivative in the reaction mixture, which was separable
by column chromatography), the same reaction of 23 resulted in a
chromatographically only partially separable mixture of endo/exo
4:1, which had to be purified by crystallization. Dimethylamino
derivatives 12, 21 and 28 were prepared by a recently described
method using dimethylamine generated in situ by microwave-
assisted decomposition of DMF.15
X
N
N
OH
OH
NH3Cl
h
OH
N
6 steps
a
c
N
OH
NH3Cl
O
O
OH
O
9
X=Cl
6
5
7
d
e
10
11
12
X=NH2
b
X=NHcPr
X=NMe2
f
O
g
X
13 X=SH
N
N
NH
O
N
N
N
NH2
OH
OH
14 X=Cl
d
e
i
15
16
17
X=NH2
X=NHcPr
X=OH
8
Scheme 1. Reagents and conditions: (a) (1) ClCOOEt, TEA, acetone, 0 °C, 1 h, (2) NaN3, H2O, 0 °C, 1 h, (3) dioxane, HCl, reflux, 5 h, 74%; (b) (1) ethyl [(2E)-3-ethoxy-2-
methylprop-2-enoyl]carbamate, dioxane, 100°, 3 h, (2) Dowex 50W (H+), dioxane, 100 °C overnight, 61%; (c) 4,6-dichloro-5-formamidopyrimidine, DIPEA, n-BuOH, MW,
160 °C, 2 h, 55%; (d) NH3, EtOH, 120 °C, 30 min, 83% for 10, 83% for 15; (e) cyclopropylamine, EtOH, MW, 140 °C, 30 min, 88% for 11, 76% for 16; (f) DMF, MW, 200 °C, 2 min,
86%; (g) thiourea, EtOH, 105 °C, overnight, 76%; (h) 2-amino-4,6-dichloro-5-formamidopyrimidine, DIPEA, n-BuOH, MW, 160 °C, 2 h, 63%; (i) TFA, H2O, 24 h, 56%.

2976
M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
10 11
8
16
a,b
a
a
a
OH
OH
O
NH2
N
N
P
O
N
RO
RO
HN
N
HN
N
N
N
RO
RO
RO
HO
HO
O
P
N
N
N
N
O
N
O
P
O
N
N
O
P
RO
P
O
O
NH2
O
O
33 R=i-Pr
34 R=H
35
36
37 R=i-Pr
38 R=H
R=i-Pr
R=H
32
b
b
b
O
O
P
O
HO
O
O
NH
O
O
P
P
O
O
HO
NHBoc
NHBoc
NH2
N
O
OH
O
O
a
d
e
c
7
41
39
40
42
Scheme 3. Reagents and conditions: (a) (t-BuO)2Mg, TsCH2P(O)(Oi-Pr)2, DMF, 60 °C, 48 h, 89% for 33, 25% for 35, 81% for 37, 58% for 40; (b) TMSBr, DCM, 24 h, 60% for 32, 95%
for 34, 92% for 36, 90% for 38; (c) Boc2O, DIPEA, DCM, overnight, 89%; (d) TFA, DCM, 2 h, 56%; (e) (1) ethyl [(2E)-3-ethoxy-2-methylprop-2-enoyl]carbamate, dioxane, 100°,
3 h, (2) Dowex 50W (H+), dioxane, 100 °C, 12 h, 43%.
Table 2
Phosphonate functionality was introduced to 10, 11 and 16
using tosylmethanphosphonate as the alkylating agent. The use
of (t-BuO)2Mg as a base and DMF as a solvent afforded significantly
better yields than the use of t-BuONa in THF (Scheme 3). Direct
alkylation of the thymine-based compound 8 afforded a product
alkylated on both the hydroxyl group of the norbornane and the
N-3 position of the thymine nucleobase. Therefore, the phospho-
nate moiety was introduced first to the Boc-protected 7 and the
thymine was constructed afterwards. An interesting feature of this
reaction is that during the Dowex 50 (H+)-catalyzed ring-closure
reaction, the phosphonate diester is converted to a free phospho-
nate 42, which was isolated on the C-18 reverse phase.
The antiviral effect of selected compounds on the replication of Coxsackievirus in
Vero cell cultures (in
l
g mLꢀ1
)
a
b
CVB3 EC50
CVB4 EC50
CC50
9
9
16
7
15
22
10
1
2
1
2
5
1
9
9
2
0
5
0
0
0
>100
>100
>100
>100
8
>100
14
18
23
25
30
39
12
9
56
12
a
50% effective concentration or compound concentration causing 50% inhibition
of the virus-induced cytopathic effect as determined by measuring the cell viability
with the colorimetric formazan-based MTS assay.
b
50% cytotoxic concentration as determined by measuring the cell viability with
the colorimetric formazan-based MTS assay.
2.2. Biological activity
The antiviral activities of all 50-nornucleoside derivatives as
well as the nucleoside phosphonates were evaluated against a
broad panel of viruses containing RNA-, DNA- and retroviruses.
In accordance with the observation of Marquez et al.,9 these com-
pounds locked in South conformation were devoid of any activity
against HIV and human herpes viruses.
Several compounds lacking the phosphonate moiety exerted
activity against feline herpes virus (Table 1). Since the activity
was observed for unphosphorylated derivatives rather than nucle-
oside phosphonates, we can speculate that it is not based on the
incorporation of these compounds into the polynucleotide chain.
The activity seems to be rather related to the 9-norbornyl-6-ami-
nopurine skeleton, with the most active compound being the ade-
nine derivative 19.
Moreover, a number of compounds exerted activity against
Coxasckieviruses B3 and B4 (Table 2). In addition, compounds 20
and 23 inhibited the replication of Varicella zoster virus in
human embryonic lung cells with EC50 = 41
l
g mLꢀ1 and
EC50 = 20 respectively. The nucleoside phosphonates
l
g mLꢀ1
,
were also screened for inhibitory activity against adenylate cyclase
toxin from Bordetella pertussis in our cell-based assay, but none of
them exerted significant a reduction of the activity at 10
concentrations.
lM
3. Conclusion
We report on an efficient synthesis of a novel series of norborn-
ane-based conformationally locked nucleoside and nucleotide ana-
logs. Firstly, we prepared a number of 50-nornucleoside derivatives
locked in South conformation via a substitution of the natural tet-
rahydrofuran sugar ring with norbornane, bearing the purine or
pyrimidine nucleobase in the bridgehead position. Secondly, we
introduced a phosphonate moiety onto the C-3 hydroxygroup,
which led to the cyclic nucleoside phosphonates, stabilized by
bicyclo[2.2.1]heptane in the desired South conformation.
Table 1
The antiviral activity of selected compounds against feline herpes virus (in
in Crandell-Rees feline kidney cell cultures
l
g mLꢀ1
)
a
b
EC50
CC50
10
19
20
26
14
7
52
51
3
2
7
1
>100
>100
>100
>100
>100
>100
27
42 15
0.22 0.01
Our observation supports the findings of Marquez et al.,9
because none of the prepared nucleoside phosphonates exerted
significant activity against any of the viruses evaluated, although
this might be caused not only by the disfavored South
conformation but also by the enhanced steric hindrance of the
inferior nucleotide side incurred by the ethylene bridge.
Ganciclovir
a
50% effective concentration or compound concentration causing 50% inhibition
of the virus-induced cytopathic effect as determined by measuring the cell viability
with the colorimetric formazan-based MTS assay.
b
50% Cytotoxic concentration as determined by measuring the cell viability with
the colorimetric formazan-based MTS assay.

M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
2977
Several compounds obtained in the first part of our study, lack-
4.2. 1-[(1R⁄,3R⁄,4R⁄)-3-Hydroxybicyclo[2.2.1]hept-1-yl]-5-
methylpyrimidine-2,4(1H,3H)-dione (8)
ing the phosphonate moiety, significantly inhibited the replication
of feline herpes virus. The most efficient compound was norborne-
3-on derivative 19 with EC50 = 7
2
l
g mLꢀ1. Since this derivative
Free amine 7 (225 mg, 1.38 mmol, amine freed from the corre-
sponding hydrochloride using Dowex 50 (H+)) was dissolved in
dioxane (20 mL); ethyl [(2E)-3-ethoxy-2-methylprop-2-enoyl]car-
bamate (305 mg, 1.5 mmol) was added, and the reaction mixture
was heated to 100 °C for 3 h. Dowex 50 (H+, 5 g) was added, and
the reaction mixture was heated to 100 °C overnight. The Dowex
resin was filtered off, the crude product was adsorbed on silica,
and purification by flash chromatography (1–5% methanol in ethyl
acetate) and crystallization from toluene afforded 8 (200 mg, 61%)
as white crystals (mp = 256–257 °C). 1H NMR (500 MHz, DMSO):
d1.22 (m, 1H, H-6endo), 1.29 (dm, 1H, Jgem = 12.8, H-3exo), 1.44
(m, 1H, H-5exo), 1.66 (tdd, 1H, Jgem = J6ex,5ex = 12.5, J6ex,1 = 5.3,
J6ex,5en = 4.2, H-6exo), 1.74 (dm, 1H, Jgem = 9.1, H-7a), 1.77 (d, 3H,
cannot be phosphorylated and incorporated into a polynucleotide
chain by viral DNA polymerases, it seems to be plausible that this
type of compounds acts via a new, as yet unknown mechanism of
action and presents the first step towards the development of a
novel type of therapeutics complementary to the current nucleo-
side-based treatment of this widespread feline infection.
4. Experimental section
The reagents and solvents were purchased and used as received,
or prepared according to published procedures. The NMR spectra
were recorded on Bruker Avance I 500 (1H at 500 MHz, 13C at
125.8 MHz) and Bruker Avance II 600 (1H at 600 MHz, 13C at
150 MHz) spectrometers using DMSO-d6 or CDCl3 as a solvent
and using the solvent signal as a reference. The chemical shifts
(d) and coupling constants (J) were expressed in ppm and Hz,
respectively. All structures were confirmed and 1H and 13C signals
were assigned by a combination of 1D and 2D NMR (H,H-COSY,
H,C-HSQC, H,C-HMBC, ROESY) techniques. Standard pulse pro-
grams from the library of the spectrometer were used; gradient
selection was utilized in the 2D experiments. The mass spectra
were measured on an LTQ Orbitrap XL (Thermo Fisher Scientific)
using electrospray ionization (ESI). The elemental analyses were
measured on Perkin Elmer CHN Analyzer 2400, Series II Sys (Perkin
Elmer, Norwolk, CT) or on SPECTRO iQ II (Spectro Analytical Instru-
ments, Germany). The melting points are uncorrected and were
determined on a Büchi Melting Point B-540 apparatus. The micro-
wave syntheses were carried out in a CEM Discover instrument
with a single-mode cavity and focused microwave heating (a
microwave power supply of 0–300 W, 1W increments, IR temper-
ature sensor, sealed-vessel mode, a pressure range of 0–20 bar, 10
or 60 mL vials). The column chromatography was performed on a
0
JCH3,6 = 1.2, CH3), 2.01–2.07 (m, 2H, H-1, H-5endo), 2.15 (dm, 1H,
Jgem = 9.1, H-7b), 2.58 (ddd, 1H, Jgem = 12.8, J3en,2 = 7.0, J3en,7a = 2.4,
H-3endo), 3.73 (m, 1H, H-2), 4.76 (d, 1H, JOH,2 = 3.6, OH), 7.51 (q,
1H, J6 ,CH3 = 1.2, H-60), 11.08 (br s, 1H, H-30). 13C NMR (125.8 MHz,
0
DMSO): d 12.18 (CH3), 24.20 (C-6), 31.41 (C-5), 38.00 (C-7), 41.53
(C-1), 44.97 (C-3), 68.97 (C-4), 72.71 (C-2), 107.82 (C-50), 139.99
(C-60), 151.06 (C-20), 164.30 (C-40). ESI MS m/z (%): 259.1 (100)
[M+H], 281.1 (55) [M+Na]; HRMS ESI (C12H17N2O3): calculated:
237.12337; found: 237.12339. For C12H16N2O3 (236.27): calcu-
lated: 61.00; C, 6.83; H, 11.86; N; found: 61.27; C, 6.98; H, 11.72;
N.
4.3. (1S⁄,2S⁄,4S⁄)-4-(6-Chloro-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (9)
A solution of 7 (2.3 g, 14 mmol), 4,6-dichloro-5-aminopyrimi-
dine (3.4 g, 21 mmol) and DIPEA (7.9 mL, 56 mmol) in n-butanol
(80 mL) was heated in a sealed microwave vessel at 160 °C for
4 h. The resulting reaction intermediate was purified by chroma-
tography on silica gel (toluene–ethyl acetate = 1:4), dissolved in a
mixture of triethyl ortoformate (300 mL) and concd HCl (4 mL)
and stirred at room temperature for 3–5 d, and then evaporated.
This oily residue was dissolved in a mixture of THF and 1M hydro-
chloric acid (1:1, 125 mL) and stirred at rt for 4 h. After neutraliza-
tion with sodium hydrogencarbonate, all volatiles were evaporated
and the product was purified by column chromatography (ethyl
acetate—toluene—acetone—ethanol 17:4:3:1) and crystallization
from a toluene–cyclohexane mixture to afford 9 (2.1 g, 55%) as
white crystals (mp = 155–157 °C). 1H NMR (500 MHz, DMSO): d
1.35 (dddd, 1H, Jgem = 12.4, J6en-5en = 9.2, J6en-5ex = 4.8, J6en-7b = 2.1,
40–60 lm silica gel using the ISCO flash chromatography system
or standard glass columns. The purity of all of the compounds pre-
pared was higher than 98% unless stated otherwise.
4.1. (1S⁄,2S⁄,4S⁄)-4-Aminobicyclo[2.2.1]heptan-2-ol
hydrochloride (7)
Triethylamine (3.2 mL, 23 mmol) and ethyl chloroformate
(1.9 mL, 20.1 mmol) were added to a solution of 6 (3 g, 19.2 mmol)
in dry acetone (50 mL) at 0 °C. After 1 h at 0 °C, a solution of
sodium azide (3.7 g, 57.6 mmol) in water (50 mL) was added. After
1 h at 0 °C, the reaction mixture was diluted with water (400 mL)
and extracted with ethyl acetate (4 ꢁ 250 mL). The combined
organic extracts were washed with satd NaHCO3 (2 ꢁ 200 mL)
and water (200 mL), dried over sodium sulfate and evaporated.
The oily residue was dissolved in dioxane (50 mL) and 2M HCl
(150 mL), heated to reflux for 5 h, evaporated and codistilled with
toluene (3 ꢁ 200 mL) to afford crude amine, which was further
purified by precipitation as hydrochloride from the ethanol–dieth-
ylether mixture to afford 7 (2.35 g, 74%) as white crystals.
H-6endo), 1.75 (dm, 1H, Jgem = 12.4, H-3exo), 1.79 (tdd, 1H, Jgem
=
J6ex-5ex = 12.3, J6ex-1 = 5.1, J6ex-5en = 4.2, H-6exo), 1.90 (m, 1H,
H-5exo), 2.01 (dddd, 1H, Jgem = 11.3, J5en-6en = 9.1, J5en-6ex = 4.1,
J5en-7b = 2.3, H-5endo), 2.05 (dm, 1H, Jgem = 9.1, H-7a), 2.19 (dm,
1H, J1-6ex = 5.0, H-1), 2.49 (m, 1H, H-7b), 2.54 (ddd, 1H, Jgem = 12.5,
J3en-2 = 6.9, J3en-7a = 2.4, H-3endo), 3.87 (m, 1H, H-2), 4.93 (d, 1H,
JOH-2 = 3.5, OH), 8.71 (s, 1H, H-80), 8.77 (s, 1H, H-20). 13C NMR
(125.8 MHz, DMSO): d 24.04 (C-6), 32.65 (C-5), 37.57 (C-7), 42.69
(C-1), 45.85 (C-3), 65.17 (C-4), 72.67 (C-2), 131.72 (C-50), 146.59
(C-80), 149.37 (C-60), 151.28 (C-20), 152.43 (C-40). ESI MS m/z (%):
265.2 (100) [M+H]. For C12H13N4OCl (262.69): calculated: 54.45;
C, 4.95; H, 21.17; N, 13.39; Cl; found: 54.49; C, 5.08; H, 21.26; N,
12.99; Cl.
1H NMR (500 MHz, DMSO):
d 1.01 (ddt, 1H, Jgem = 8.9,
J7a-3en = 2.5, J7a-1 = J7a-2 = 1.5, H-7a), 1.05–1.12 (m, 2H, H-5endo,
H-6endo), 1.16 (ddd, 1H, Jgem = 12.4, J3ex-5ex = 3.6, J3ex-2 = 2.6,
H-3exo), 1.27 (m, 1H, H-5exo), 1.43–1.55 (m, 2H, H-6exo, H-7b),
1.62 (ddd, 1H, Jgem = 12.4, J3en-2 = 7.0, J3en-7a = 2.4, H-3endo), 1.84
(dm, 1H, J1-6ex = 5.1, H-1), 3.62 (dm, 1H, J2-3en = 7.0, H-2). 13C
NMR (125.8 MHz, DMSO): d 25.22 (C-6), 35.37 (C-5), 41.75 (C-7),
43.63 (C-1), 48.87 (C-3), 61.41 (C-4), 73.80 (C-2). HRMS
(C7H14NO): calculated: 128.1075; found: 128.1070 (M+H).
4.4. (1R⁄,2R⁄,4R⁄)-4-(2-Amino-6-chloro-9H-purin-9-yl)
bicyclo[2.2.1]heptan-2-ol (14)
2-Amino-4,6-dichloro-5-formamidopyrimidine (2.4 g, 11.5 mmol)
and DIPEA (5 mL, 28.8 mmol) were added to a solution of the 7 (2.2
g, 9.6 mmol) in n-butanol (40 mL), and the reaction mixture was

2978
M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
heated in a sealed microwave vessel at 160 °C for 2 h. Flash chro-
matography (2–10% methanol in ethyl acetate) followed by crys-
tallization from toluene afforded 14 (1.4 g, 63%) as off-white
crystals (mp = 218–219 °C (decomp.)). 1H NMR (500 MHz, DMSO):
d 1.29 (m, 1H, H-6endo), 1.62 (dm, 1H, Jgem = 12.5, H-3exo), 1.70–
1.83 (m, 2H, H-5exo, H-6exo), 1.93 (m, 1H, H-5endo), 2.01 (dm,
1H, Jgem = 9.1, H-7a), 2.15 (m, 1H, H-1); 2.35 (dm, 1H, Jgem = 9.2,
H-7b), 2.51 (ddd, 1H, Jgem = 12.5, J3en,2 = 6.9, J3en,7 = 2.6, H-3endo),
3.82 (m, 1H, H-2), 4.87 (t, 1H, JOH-CH2 = 3.5, OH), 6.79 (br s, 2H,
NH2), 8.11 (s, 1H, H-80). 13C NMR (125.8 MHz, DMSO): d 24.13 (C-
6), 32.41 (C-5), 37.38 (C-7), 42.64 (C-1), 45.79 (C-3), 64.41 (C-4),
72.69 (C-2), 124.26 (C-50), 142.21 (C-80), 149.66 (C-60), 154.65 (C-
40), 159.49 (C-20). ESI MS m/z (%): 280.1 (97) [M+H], 302.1 (100)
[M+Na]; HRMS ESI (C12H14N5OCl): calculated: 280.09596, found:
280.09595. For C12H14ClN5O (279.73): calculated: 51.52; C, 5.04;
H, 12.67; Cl, 25.04; N; found: 51.55; C, 5.05; H, 12.59; Cl, 25.10; N.
A saturated solution of ammonium chloride (30 mL) was added
and the product was extracted with ethyl acetate (5 ꢁ 30 mL).
The combined organic extracts were dried over sodium sulfate
and chromatographed on silica gel (ethyl acetate—toluene—ace-
tone—ethanol 17:4:3:1) to afford 25 (540 mg, 89%) as white pow-
der. For analytical purposes, 100 mg of the product were
crystallized from a toluene–cyclohexane mixture (white crystals,
mp = 154–155 °C). 1H NMR (500 MHz, DMSO): d 1.65 (m, 1H, H-
6exo), 1.80 (dm, 1H, Jgem = 12.2, H-3endo), 1.97 (m, 1H, H-5exo),
2.11–2.21 (m, 4H, H-5endo, H-6endo, H-7), 2.29 (m, 1H, H-1),
2.31 (ddd, 1H, Jgem = 12.3, J3ex-2 = 10.4, J3ex-5ex = 3.8, H-3exo), 4.28
(m, 1H, H-2), 4.99 (d, 1H, JOH-2 = 4.1, OH), 8.66 (s, 1H, H-80), 8.76
(s, 1H, H-20). 13C NMR (125.8 MHz, DMSO): d 20.81 (C-6), 34.16
(C-5), 40.15 (C-7), 41.00 (C-1), 43.61 (C-3), 66.11 (C-4), 69.74 (C-
2), 131.71 (C-50), 146.37 (C-80), 149.34 (C-60), 151.26 (C-20),
152.32 (C-40). ESI MS m/z (%): 263.2 (100) [M+H]. For C12H13N4OCl
(262.69): calculated: 54.45; C, 4.95; H, 21.17; N, 13.39; Cl; found:
54.28; C, 5.02; H, 21.10; N, 13.39; Cl.
4.5. (1S⁄,4S⁄)-4-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.1]heptan-2-
one (18)
4.8. (1R⁄,2S⁄,4R⁄)-4-(2-Amino-6-chloro-9H-purin-9-yl)bicyclo
A solution of 9 (1 g, 3.8 mmol) in dichlormethane (50 mL) was
added dropwise to a suspension of PDC (2.84 g, 7.6 mmol) and
crushed molecular sieves (3 g) in dichlormethane (50 mL). The
reaction mixture was stirred at rt for 7 d, the solid parts were fil-
tered off and thoroughly washed with chloroform. The resulting
brown oil was adsorbed on silica gel and chromatographed on a
short column of silica (ethyl acetate—toluene—acetone—ethanol
17:4:3:1). Crystallization from a water–methanol mixture afforded
18 (830 mg, 82%) as white crystals (mp = 167–168 °C).
[2.2.1]heptan-2-ol (30)
NaBH4 (39 mg, 1 mmol) was added portionwise to a solution of
23 (470 mg, 1.7 mmol) in dry methanol (30 mL) at 0 °C and the
reaction mixture was stirred at this temperature for 5 h. A satu-
rated solution of ammonium chloride (30 mL) and water
(100 mL) was added and the product was extracted into ethyl ace-
tate (5 ꢁ 50 mL). The organic extracts were combined and dried
with sodium sulfate. Flash chromatography (1–10% methanol in
ethyl acetate) and multiple crystallization from a toluene–ethyl
acetate mixture afforded 30 (430 mg, 51%) as white powder
(mp = 213–214 °C). 1H NMR (500 MHz, DMSO): d 1.59 (m, 1H, H-
6exo), 1.74 (dm, 1H, Jgem = 12.3, H-3endo), 1.89 (m, 1H, H-5exo),
2.07–2.15 (m, 4H, H-5endo, H-6endo, H-7), 2.21 (ddd, 1H,
Jgem = 12.3, J3ex,2 = 10.4, J3ex,5ex = 3.7, H-3exo), 2.23 (m, 1H, H-1),
4.24 (m, 1H, H-2), 4.93 (d, 1H, JOH-2 = 3.9, OH), 6.81 (br s, 2H,
NH2), 8.06 (s, 1H, H-80). 13C NMR (125.8 MHz, DMSO): d 24.74 (C-
6), 33.91 (C-5), 40.00 (C-7), 40.93 (C-1), 43.37 (C-3), 65.33 (C-4),
69.76 (C-2), 124.25 (C-50), 141.98 (C-80), 149.60 (C-60), 154.53 (C-
40), 159.46 (C-20). ESI MS m/z (%): 280.1 (73) [M+H], 302.1 (100)
[M+Na]; HRMS ESI (C12H14ON5ClNa): calculated: 302.07791;
found: 302.07789.
1H NMR (500 MHz, DMSO): d 1.58 (m, 1H, H-6endo), 2.12 (tt,
1H, Jgem = J6ex-5ex = 12.6, J6ex-1 = J6ex-5en = 4.9, H-6exo), 2.27 (m, 1H,
H-5exo), 2.34 (dddd, 1H, Jgem = 11.7, J5en-6en = 9.0, J5en-6ex = 4.8,
J5en-7 = 2.0, H-5endo), 2.52–2.60 (m, 2H, H-7), 2.79 (dm, 1H,
J1-6ex = 5.0, H-1), 2.85–2.86 (m, 2H, H-3), 8.77 (s, 1H, H-80), 8.80
(s, 1H, H-20). 13C NMR (125.8 MHz, DMSO): d 23.31 (C-6), 31.68
(C-5), 39.95 (C-7), 48.60 (C-3), 49.00 (C-1), 63.47 (C-4), 131.74
(C-50), 146.37 (C-80), 149.49 (C-60), 151.48 (C-20), 152.35 (C-40)
211.07 (C-2). ESI MS m/z (%): 263.1 (100) [M+H], 285.1 (4)
[M+Na]. For C12H11N4OCl (262.69): calculated: 54.87; C, 4.22; H,
21.33; N, 13.50; Cl; found: 54.70; C, 4.21; H, 20.99; N, 13.39; Cl.
4.6. (1R⁄,4R⁄)-4-(2-Amino-6-chloro-9H-purin-9-yl)bicyclo[2.2.1]
heptan-2-one (23)
4.9. Microwave-assisted ammonolysis of the C-6 chlorine atom
of the purine nucleobase (compounds 10, 15, 19 and 26)
A solution of 14 (1.11 g, 4 mmol) in DMF (10 mL) was added
dropwise to a solution of PDC (3 g, 8 mmol) in DMF (70 mL). The
reaction mixture was stirred at rt for 12 h, the volatiles were
evaporated and the product was purified by flash chromatography
(1–3% methanol in ethyl acetate). Crystallization from toluene
afforded 23 (620 mg, 56%) as white crystals (mp = 209–210 °C).
1H NMR (500 MHz, DMSO): d 1.53 (m, 1H, H-6endo), 2.07 (m, 1H,
H-6exo), 2.18–2.25 (m, 2H, H-5), 2.44 (ddd, 1H, Jgem = 9.8,
J7b,3en = 3.9, J7b,1 = 1.3, H-7b), 2.49 (m, 1H, H-7a), 2.71–2.84 (m,
3H, H-1, H-3), 6.90 (br s, 2H, NH2), 8.19 (s, 1H, H-80). 13C NMR
(125.8 MHz, DMSO): d 23.27 (C-6), 31.35 (C-5), 39.7 (C-7), 48.25
(C-3), 48.96 (C-1), 62.78 (C-4), 124.17 (C-50), 141.83 (C-80),
149.80 (C-60), 154.50 (C-40), 159.59 (C-20), 211.54 (C-2). ESI MS
m/z (%): 278.1 (43) [M+H], 300.0 (100) [M+Na]; HRMS ESI
(C12H13ON5Cl): calculated: 278.08031; found: 278.08041.
A solution of a 6-chloropurine or 2-amino-6-chloropurine
derivative (up to 1 mmol) in ethanolic ammonia (3.5 M, 5 mL)
was heated in a microwave reactor at 120 °C for 20–40 min (TLC
determination of the reaction completion). The volatiles were
evaporated and the crude compound was adsorbed on silica gel
and purified by flash chromatography and subsequent crystalliza-
tion from aqueous methanol.
4.9.1. (1S⁄,2S⁄,4S⁄)-4-(6-Amino-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (10)
From 9 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—
acetone—ethanol—water 100:15:6:4; crystallization from water;
yield: 100 mg, 83%, colorless needles (mp = 253–254 °C). 1H NMR
(500 MHz, DMSO): d 1.31 (dddd, 1H, Jgem = 12.4, J6en-5en = 9.1,
J6en-5ex = 4.7, J6en-7b = 2.1, H-6endo), 1.69 (dm, 1H, Jgem = 12.4,
H-3exo), 1.75 (tdd, 1H, Jgem = J6ex-5ex = 12.3, J6ex-1 = 5.0,
J6ex-5en = 4.2, H-6exo), 1.85 (m, 1H, H-5exo), 1.94 (dddd, 1H,
Jgem = 11.3, J5en-6en = 9.1, J5en-6ex = 4.1, J5en-7b = 2.2, H-5endo), 2.00
(dm, 1H, Jgem = 9.1, H-7a), 2.15 (dm, 1H, J1-6ex = 5.0, H-1), 2.39
(dq, 1H, Jgem = 9.1, J7b-1 = J7b-5en = J7b-6en = 2.0, H-7b), 2.49 (ddd,
4.7. (1S⁄,2R⁄,4S⁄)-4-(6-Chloro-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (25)
NaBH4 (52 mg, 1.4 mmol) was added portionwise to a solution
of 18 (600 mg, 2.3 mmol) in dry methanol (15 mL) at 0 °C and
the reaction mixture was stirred at this temperature for one hour.

M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
2979
1H, Jgem = 12.6, J3en-2 = 6.8, J3en-7a = 2.4, H-3endo), 3.84 (m, 1H, H-2),
4.88 (d, 1H, JOH-2 = 3.5, OH), 7.19 (br s, 2H, NH2), 8.116 and 8.120 (s,
2H, H-20, H-80). 13C NMR (125.8 MHz, DMSO): d 24.22 (C-6), 32.73
(C-5), 37.61 (C-7), 42.75 (C-1), 46.02 (C-3), 64.38 (C-4), 72.81 (C-2),
1 mmol of substrate) was heated in a microwave reactor at
140 °C for 10–40 min (TLC determination of the reaction comple-
tion). The volatiles were evaporated and the crude product was
adsorbed on silica and purified by column chromatography and
crystallization.
119.82
(C-50),
139.74
(C-80),
150.24
(C-40),
152.27
(C-20), 156.31 (C-60). ESI MS m/z (%): 246.2 (100) [M+H], 268.1
(21) [M+Na]. For C12H15N5O (245.28): calculated: 58.76; C, 6.16;
H, 28.55; N; found: 58.48; C, 6.17; H, 28.28; N.
4.10.1. (1S⁄,2S⁄,4S⁄)-4-[6-(Cyclopropylamino)-9H-purin-9-
yl]bicyclo [2.2.1]heptan-2-ol (11)
From 9 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—tolu-
ene—acetone—ethanol 17:4:3:1; crystallization from a toluene–
cyclohexane mixture; yield: 123 mg, 88%, white crystals
(mp = 155–156 °C). 1H NMR (500 MHz, DMSO): d 0.60 and 0.71
(m, 4H, CH2-cyclop), 1.31 (dddd, 1H, Jgem = 12.3, J6en-5en = 9.0,
J6en-5ex = 4.8, J6en-7b = 2.0, H-6endo), 1.69 (dm, 1H, Jgem = 12.5,
4.9.2. (1S⁄,2S⁄,4S⁄)-4-(2,6-Diamino-9H-purin-9-yl)bicyclo[2.2.1]
heptan-2-ol (15)
From 14 (220 mg, 0.79 mmol); mobile phase: 10–15% methanol
in ethyl acetate; crystallization from a water–methanol mixture;
yield: 170 mg, 83%, pale-orange crystals (mp = 295–296 °C). 1H
NMR (500 MHz, DMSO): d 1.27 (m, 1H, H-6endo), 1.59 (dm, 1H,
Jgem = 12.6, H-3exo), 1.68–1.81 (m, 2H, H-5exo, H-6exo), 1.89 (m,
1H, H-5endo), 2.00 (dm, 1H, Jgem = 9.1, H-7a), 2.12 (m, 1H, H-1);
2.28 (dm, 1H, Jgem = 9.1, H-7b), 2.49 (m, 1H, H-3endo), 3.80 (m,
1H, H-2), 4.82 (m, 1H, OH), 5.63 (br s, 2H, 20-NH2), 6.60 (br s, 2H,
60-NH2), 7.68 (s, 1H, H-80). 13C NMR (125.8 MHz, DMSO): d 24.27
(C-6), 32.51 (C-5), 37.44 (C-7), 42.66 (C-1), 45.98 (C-3), 63.82
(C-4), 72.79 (C-2), 114.28 (C-50), 136.27 (C-80), 152.53 (C-40),
156.33 (C-60), 159.91 (C-20). ESI MS m/z (%): 261.3 (100) [M+H],
283.3 (14) [M+Na]; HRMS ESI (C12H17ON6): calculated:
261.14584; found: 261.14578. For C12H16N6O (260.30): calculated:
55.37; C, 6.20; H, 32.29; N; found: 55.21; C, 6.29; H, 32.59; N.
H-3exo), 1.75 (tdd, 1H, Jgem = J6ex-5ex = 12.3, J6ex-1 = 5.0, J6ex-5en
=
4.2, H-6exo), 1.85 (m, 1H, H-5exo), 1.94 (dddd, 1H, Jgem = 11.3,
J5en-6en = 9.2, J5en-6ex = 4.1, J5en-7b = 2.2, H-5endo), 2.00 (dm, 1H,
Jgem = 9.1, H-7a), 2.15 (dm, 1H, J1-6ex = 4.9, H-1), 2.39 (dm, 1H,
Jgem = 9.1, H-7b), 2.50 (ddd, 1H, Jgem = 12.5, J3en-2 = 6.9, J3en-7a
= 2.4, H-3endo), 2.99 (br s, 1H, CH-cyclop), 3.84 (m, 1H, H-2),
4.88 (d, 1H, JOH-2 = 3.5, OH), 7.86 (br s, 1H, NH), 8.12 (s, 1H, H-80),
8.22 (s, 1H, H-20). 13C NMR (125.8 MHz, DMSO): d 6.61 (CH2-
cyclop), 24.23 (C-6), 32.75 (C-5), 37.63 (C-7), 42.75 (C-1), 46.03
(C-3), 64.40 (C-4), 72.81 (C-2), 120.20 (C-50), 139.59 (C-80), 149.69
(C-40), 152.18 (C-20), 155.88 (C-60). ESI MS m/z (%): 286.2 (100)
[M+H], 308.2 (12) [M+Na]. For C15H19N5O (285.16): calculated:
63.14; C, 6.71; H, 24.54; N; found: 62.86; C, 6.83; H, 24.24; N.
4.9.3. (1S⁄,4S⁄)-4-(6-Amino-9H-purin-9-yl)bicyclo[2.2.1]heptan-
2-one (19)
4.10.2. (1R⁄,2R⁄,4R⁄)-4-[2-Amino-6-(cyclopropylamino)-9H-
purin-9-yl]bicyclo[2.2.1]heptan-2-ol (16)
From 18 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—ace-
tone—ethanol—water 100:15:6:4; crystallization from a water–
methanol mixture; yield: 93 mg, 77%, colorless crystals
(mp = 239–240 °C). 1H NMR (500 MHz, DMSO): d 1.54 (m, 1H, H-
6endo), 2.09 (m, 1H, H-6exo), 2.21–2.30 (m, 2H, H-5), 2.46–2.52
(m, 2H, H-7), 2.71 (m, 1H, H-1), 2.75–2.85 (m, 2H, H-3), 7.25 (br
s, 2H, NH2), 8.14 (s, 1H, H-20), 8.19 (s, 1H, H-80).13C NMR
(125.8 MHz, DMSO): d 23.40 (C-6), 31.64 (C-5), 39.99 (C-7), 48.64
(C-3), 49.08 (C-1), 62.79 (C-4) 119.73 (C-50), 139.44 (C-80), 150.08
(C-40), 152.44 (C-20), 156.35 (C-60), 211.68 (C-2). ESI MS m/z (%):
244.1 (100) [M+H]. For C12H13N5O (243.26): calculated: 59.25; C,
5.39; H, 28.79; N; found: 58.96; C, 5.35; H, 28.45; N.
From 14 (490 mg, 1.75 mmol). The poorly soluble product was
filtered off and thoroughly washed with water and methanol.
Yield: 400 mg, 76%, pink crystals (mp = 258 °C (decomp.)). 1H
NMR (500 MHz, DMSO): d 0.57 and 0.65 (m, 4H, CH2-cyclop),
1.27 (m, 1H, H-6endo), 1.58 (dm, 1H, Jgem = 12.5, H-3exo), 1.68–
1.80 (m, 2H, H-5exo, H-6exo), 1.89 (m, 1H, H-5endo), 2.00 (m,
1H, Jgem = 9.1, H-7a), 2.12 (m, 1H, H-1), 2.28 (dm, 1H, Jgem = 9.2,
H-7b), 2.50 (m, 1H, H-3endo), 3.01 (br s, 1H, CH-cyclop), 3.80 (m,
1H, H-2), 4.81 (d, 1H, JOH,2 = 3.6, OH), 5.69 (br s, 2H, NH2), 7.21
(br s, 1H, NH), 7.66 (s, 1H, H-80). 13C NMR (125.8 MHz, DMSO): d
6.58 (CH2-cyclop), 24.27 (C-6), 32.51 (C-5), 37.44 (C-7), 42.65
(C-1), 45.98 (C-3), 63.80 (C-4), 72.78 (C-2), 114.54 (C-50), 135.96
(C-80), 152.1 (C-40), 156.08 (C-60), 159.82 (C-20). ESI MS m/z (%):
301.3 (100) [M+H], 323.3 (10) [M+Na]; HRMS ESI (C15H21ON6): cal-
culated: 301.17714; found: 301.17697. For C15H20N6O (300.36):
calculated: 59.98; C, 6.71; H, 27.98; N; found: 60.11; C, 6.80; H,
27.80; N.
4.9.4. (1S⁄,2R⁄,4S⁄)-4-(6-Amino-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (26)
From 25 (110 mg, 0.4 mmol); mobile phase: ethyl acetate—
acetone—ethanol—water 100:15:6:4; crystallization from water;
yield: 71 mg, 70%, colorless crystals (mp = 252–253 °C). 1H NMR
(500 MHz, DMSO):
d 1.61 (m, 1H, H-6exo), 1.73 (dm, 1H,
Jgem = 12.3, H-3endo), 1.93 (m, 1H, H-5exo), 2.08–2.16 (m, 4H,
H-5endo, H-6endo, H-7), 2.24 (m, 1H, H-1), 2.58 (ddd, 1H,
Jgem = 12.3, J3ex-2 = 10.5, J3ex-5ex = 3.1, H-3exo), 4.26 (m, 1H, H-2),
4.93 (d, 1H, JOH-2 = 4.1, OH), 7.15 (br s, 2H, NH2), 8.06 (s, 1H,
H-80), 8.11 (s, 1H, H-20). 13C NMR (125.8 MHz, DMSO): d 20.84
(C-6), 34.25 (C-5), 40.23 (C-7), 41.07 (C-1), 43.69 (C-3), 65.30
(C-4), 69.89 (C-2), 119.81 (C-50), 139.50 (C-80), 150.11 (C-40),
152.23 (C-20), 156.28 (C-60). NegESI MS m/z (%): 244.2 (100)
[MꢀH]. For C12H15N5O (245.28): calculated: 58.76; C, 6.16; H,
28.55; N; found: 58.39; C, 6.25; H, 28.65; N.
4.10.3. (1S⁄,4S⁄)-4-[6-(Cyclopropylamino)-9H-purin-9-
yl]bicyclo[2.2.1] heptan-2-one (20)
From 18 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—tol-
uene—acetone—ethanol 17:4:3:1; crystallization from a toluene-
cyclohexane mixture; yield: 104 mg, 77%, white crystals
(mp = 173–174 °C). 1H NMR (500 MHz, DMSO): d 0.61 and 0.72
(m, 4H, CH2-cyclop), 1.55 (m, 1H, H-6endo), 2.10 (m, 1H, H-
6exo), 2.20–2.30 (m, 2H, H-5), 2.47–2.52 (m, 2H, H-7), 2.72 (dm,
1H, J1-6ex = 5.2, H-1), 2.75–2.85 (m, 2H, H-3), 3.02 (br s, 1H, CH-cyc-
lop), 7.91 (br s, 1H, NH), 8.19 (s, 1H, H-80), 8.24 (s, 1H, H-20). 13C
NMR (125.8 MHz, DMSO): d 6.58 (CH2-cykclop), 23.41 (C-6),
31.65 (C-5), 39.99 (C-7), 48.66 (C-3), 49.07 (C-1), 62.80 (C-4),
120.12 (C-50), 139.29 (C-80), 149.63 (C-40), 152.34 (C-20), 155.85
(C-60), 211.67 (C-2). ESI MS m/z (%): 284.2 (100) [M+H]. For
4.10. Microwave-assisted nucleophilic displacement of the C-6
chlorine atom of the purine nucleobase with cyclopropylamine
(compounds 11, 16, 20 and 27)
C
15H17N5O (283.32): calculated: 63.59; C, 6.05; H, 24.72; N; found:
A solution of a 6-chloropurine or 2-amino-6-chloropurine
derivative and cyclopropylamine (10 equiv) in ethanol (5 mL per
63.33; C, 6.04; H, 24.36; N.

2980
M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
4.10.4. (1S⁄,2R⁄,4S⁄)-4-[6-(Cyclopropylamino)-9H-purin-9-
yl]bicyclo [2.2.1]heptan-2-ol (27)
4.11.3. (1S⁄,2R⁄,4S⁄)-4-[6-(Dimethylamino)-9H-purin-9-
yl]bicyclo[2.2.1] heptan-2-ol (28)
From 25 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—tol-
uene—acetone—ethanol 17:4:3:1; crystallization from a toluene–
cyclohexane mixture; yield: 117 mg, 85%, white crystals
(mp = 201–202 °C). 1H NMR (500 MHz, DMSO): d 0.59 (m, 2H,
CH2-cyclop), 0.70 (m, 2H, CH2-cyclop), 1.61 (m, 1H, H-6exo), 1.73
(dm, 1H, Jgem = 12.4, H-3endo), 1.93 (m, 1H, H-5exo), 2.08–2.16
(m, 4H, H-5endo, H-6endo, H-7), 2.22–2.30 (m, 2H, H-3exo, H-1),
3.00 (br s, 1H, CH-cyclop), 4.25 (m, 1H, H-2), 4.94 (d, 1H, JOH-2
= 4.1, OH), 7.85 (br s, 1H, NH), 8.07 (s, 1H, H-80), 8.22 (br s, 1H,
H-20). 13C NMR (125.8 MHz, DMSO): d 6.57 (CH2-cyclop), 20.86
(C-6), 34.27 (C-5), 40.24 (C-7), 41.08 (C-1), 43.71 (C-3), 65.32
(C-4), 69.90 (C-2), 120.21 (C-50), 139.35 (C-80), 149.49 (C-40),
152.15 (C-20), 155.83 (C-60). ESI MS m/z (%): 286.3 (100) [M+H].
For C15H19N5O (285.16): calculated: 63.14; C, 6.71; H, 24.54; N;
found: 63.12; C, 6.74; H, 24.69; N.
From 25 (110 mg, 0.4 mmol mmol); mobile phase: ethyl ace-
tate—toluene—acetone—ethanol 17:4:3:1; crystallization from a
toluene–cyclohexane mixture; yield: 87 mg, 77%, white crystals
(mp = 156–157 °C). 1H NMR (500 MHz, DMSO): d 1.60 (m, 1H,
H-6exo), 1.74 (dm, 1H, Jgem = 12.4, H-3endo), 1.91 (m, 1H,
H-5exo), 2.08–2.16 (m, 4H, H-5endo, H-6endo, H-7), 2.22–2.30
(m, 2H, H-1, H-3exo), 3.44 (br s, 6H, CH3), 4.25 (m, 1H, H-2), 4.94
(d, 1H, JOH-2 = 4.0, OH), 8.07 (s, 1H, H-80), 8.19 (s, 1H, H-20). 13C
NMR (125.8 MHz, DMSO): d 20.84 (C-6), 34.15 (C-5), 40.17 (C-7),
41.03 (C-1), 43.61 (C-3), 65.35 (C-4), 69.88 (C-2), 120.38 (C-50),
138.38 (C-80), 150.95 (C-40), 151.55 (C-20), 154.54 (C-60). ESI MS
m/z (%): 274.3 (100) [M+H]. For C14H19N5O (273.33): calculated:
61.52; C, 7.01; H, 25.62; N; found: 61.75; C, 7.05; H, 25.69; N.
4.12. Nucleophilic displacement of a C-6 chlorine atom of the
purine nucleobase with a sulfanyl group (compounds 13, 22,
and 29)
4.11. Microwave-assisted nucleophilic displacement of a C-6
chlorine atom of the purine nucleobase with in-situ generated
dimethylamine (compounds 12, 21, and 28)
A solution of a 6-chloropurine derivative (0.5 mmol) and thio-
urea (0.6 mmol) in dry ethanol (4 mL) was heated in a pressure
vessel at 105 °C overnight. The poorly soluble product was col-
lected on a paper filter and washed thoroughly with ethanol and
diethylether.
A solution of substrate (0.5 mmol) in DMF (3 mL) was sub-
jected to microwave irradiation (sealed vessel, 200 °C, 2 min).
The volatiles were evaporated, and the crude product was
adsorbed on silica and purified by column chromatography
and crystallization.
4.12.1. (1S⁄,2S⁄,4S⁄)-4-(6-Sulfanyl-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (13)
From 9 (130 mg, 0.5 mmol); yield: 98 mg, 76%, white powder
(mp >320 °C (decomp.)). 1H NMR (500 MHz, DMSO): d 1.31 (dddd,
1H, Jgem = 12.1, J6en-5en = 8.8, J6en-5ex = 4.6, J6en-7b = 2.1, H-6endo),
4.11.1. (1S⁄,2S⁄,4S⁄)-4-[6-(Dimethylamino)-9H-purin-9-
yl]bicyclo[2.2.1] heptan-2-ol (12)
From 9 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—tolu-
ene—acetone—ethanol 17:4:3:1; crystallization from a toluene—
cyclohexane mixture; yield: 116 mg, 86%, white crystals
(mp = 143–144 °C). 1H NMR (500 MHz, DMSO): d 1.31 (dddd, 1H,
Jgem = 12.3, J6en-5en = 9.2, J6en-5ex = 4.7, J6en-7b = 2.1, H-6endo), 1.68
(dm, 1H, Jgem = 12.4, H-3exo), 1.75 (tdd, 1H, Jgem = J6ex-5ex = 12.3,
J6ex-1 = 5.1, J6ex-5en = 4.0, H-6exo), 1.84 (m, 1H, H-5exo), 1.95 (dddd,
1H, Jgem = 11.3, J5en-6en = 9.4, J5en-6ex = 4.1, J5en-7b = 2.2, H-5endo),
2.01 (dm, 1H, Jgem = 9.0, H-7a), 2.15 (dm, 1H, J1-6ex = 5.0, H-1),
2.39 (dq, 1H, Jgem = 9.1, J7b-1 = J7b-5en = J7b-6en = 2.0, H-7b), 2.51
(ddd, 1H, Jgem = 12.4, J3en-2 = 6.9, J3en-7a = 2.4, H-3endo), 3.44 (br s,
6H, CH3), 3.84 (m, 1H, H-2), 4.88 (d, 1H, JOH-2 = 3.6, OH), 8.12
(s, 1H, H-80), 8.19 (s, 1H, H-20). 13C NMR (125.8 MHz, DMSO):
1.67 (dm, 1H, Jgem = 12.5, H-3exo), 1.75 (tdd, 1H, Jgem = J6ex-5ex
=
12.1, J6ex-1 = 5.1, J6ex-5en = 4.0, H-6exo), 1.83 (m, 1H, H-5exo), 1.95
(m, 1H, H-5endo), 1.98 (dm, 1H, Jgem = 9.1, H-7a), 2.16 (dm, 1H,
J1-6ex = 5.0, H-1), 2.40 (dq, 1H, Jgem = 9.2, J7b-1 = J7b-5en = J7b-6en
=
2.0, H-7b), 2.49 (ddd, 1H, Jgem = 12.4, J3en-2 = 6.8, J3en-7a = 2.4,
H-3endo), 3.84 (dm, 1H, J2-3en = 6.5, H-2), 4.92 (br s, 1H, OH),
8.18 (d, 1H, J2 -1 = 3.8, H-20), 8.29 (s, 1H, H-80), 13.74 (br s, 1H,
H-10).13C NMR (125.8 MHz, DMSO): d 24.14 (C-6), 32.94 (C-5),
37.70 (C-7), 42.71 (C-1), 46.13 (C-3), 64.87 (C-4), 72.73 (C-2),
136.05 (C-50), 142.17 (C-80), 144.47 (C-20), 144.78 (C-40), 176.07
(C-60). ESI MS m/z (%): 263.1 (12) [M+H], 285.1 (100) [M+Na]. For
C12H14N4OS (262.33): calculated: 54.94; C, 5.38; H, 21.36; N,
12.22; S; found: 54.64; C, 5.37; H, 21.09; N, 12.12; S.
0
0
d
24.21 (C-6), 32.59 (C-5), 37.54 (C-7), 42.69 (C-1), 45.91
(C-3), 64.42 (C-4), 72.78 (C-2), 120.36 (C-50), 138.58 (C-80),
4.12.2. (1S⁄,4S⁄)-4-(6-Sulfanyl-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-one (22)
151.07 (C-40), 151.55 (C-20), 154.53 (C-60). ESI MS m/z (%):
274.2 (100) [M+H], 296.1 (43) [M+Na]. For
C14H19N5O
From 18 (130 mg, 0.5 mmol); yield: 115 mg, 89%, white powder
(mp >320 °C (decomp.)). 1H NMR (500 MHz, DMSO): d 1.55 (m, 1H,
H-6endo), 2.09 (tt, 1H, Jgem = J6ex-5ex = 12.5, J6ex-1 = J6ex-5en = 4.9,
H-6exo), 2.20 (m, 1H, H-5exo), 2.27 (m, 1H, Jgem = 11.7,
J5en-6en = 9.0, J5en-6ex = 4.8, J5en-7 = 1.9, H-5endo), 2.46–2.50 (m, 2H,
H-7), 2.73 (dm, 1H, J1-6ex = 4.9, H-1), 2.78–2.79 (m, 2H, H-3), 8.19
(273.33): calculated: 61.52; C, 7.01; H, 25.62; N; found:
61.43; C, 7.04; H, 25.26; N.
4.11.2. (1S⁄,4S⁄)-4-[6-(Dimethylamino)-9H-purin-9-
yl]bicyclo[2.2.1] heptan-2-one (21)
(bd, 1H, J2 -1 = 1.8, H-20), 8.34 (s, 1H, H-80), 13.77 (br s, 1H, H-10).
13C NMR (125.8 MHz, DMSO): d 23.37 (C-6), 31.92 (C-5), 40.05
(C-7), 48.78 (C-3), 49.00 (C-1), 63.18 (C-4), 136.04 (C-50), 141.82
(C-80), 144.64 (C-40), 144.72 (C-20), 176.20 (C-60), 211.22 (C-2). ESI
MS m/z (%): 261.1 (18) [M+H], 283.1 (100) [M+Na]. For C12H12N4OS
(260.31): calculated: 55.37; C, 4.65; H, 21.52; N, 12.32; S; found:
55.16; C, 4.66; H, 21.17; N, 12.39; S.
From 18 (130 mg, 0.5 mmol); mobile phase: ethyl acetate—tol-
uene—acetone—ethanol 17:4:3:1; crystallization from a toluene–
cyclohexane mixture; yield: 116 mg, 86%, white crystals
(mp = 160–161 °C). 1H NMR (500 MHz, DMSO): d 1.55 (m, 1H,
H-6endo), 2.10 (m, 1H, H-6exo), 2.19–2.31 (m, 2H, H-5), 2.28 (m,
2H, H-3), 2.49 (m, 2H, H-7), 2.71 (dm, 1H, J1-6ex = 5.1, H-1), 3.46
(br s, 6H, CH3), 8.19 (s, 1H, H-80), 8.22 (s, 1H, H-20). 13C NMR
(125.8 MHz, DMSO): d 23.40 (C-6), 31.53 (C-5), 38.16 (CH3),
39.91 (C-7), 48.55 (C-3), 49.03 (C-1), 62.82 (C-4), 120.28 (C-50),
138.29 (C-80), 150.91 (C-40), 151.73 (C-20), 154.55 (C-60), 211.65
0
0
4.12.3. (1S⁄,2R⁄,4S⁄)-4-(6-Sulfanyl-9H-purin-9-
yl)bicyclo[2.2.1]heptan-2-ol (29)
(C-2). ESI MS m/z (%): 272.2 (100) [M+H]. For
(271.31): calculated: 61.98; C, 6.32; H, 25.81; N; found: 61.55; C,
6.33; H, 25.52; N.
C14H17N5O
From 25 (110 mg, 0.4 mmol); yield: 74 mg, 68%, white powder
(mp >320 °C (decomp.)). 1H NMR (500 MHz, DMSO): d 1.62 (m,
1H, H-6exo), 1.75 (m, 1H, Jgem = 12.4, H-3endo), 1.90 (m, 1H,

M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
2981
H-5exo), 2.07–2.16 (m, 4H, H-5endo, H-6endo, H-7), 2.20–2.26 (m,
2H, H-1, H-3exo), 4.25 (m, 1H, H-2), 4.97 (br s, 1H, OH), 8.17 (s, 1H,
H-20), 8.23 (s, 1H, H-80), 13.70 (br s, 1H, NH). 13C NMR (125.8 MHz,
DMSO): d 20.88 (C-6), 34.44 (C-5), 40.29 (C-7), 41.02 (C-1), 43.89
(C-3), 65.81 (C-4), 69.80 (C-2), 136.05 (C-50), 141.90 (C-80),
144.46 (C-20), 144.67 (C-40), 176.07 (C-60). EI MS m/z (%): 262.1
(100) [M]. For C12H14N4OS (262.33): calculated: 54.94; C, 5.38; H,
21.36; N, 12.22; S; found: 54.61; C, 5.40; H, 21.60; N, 12.26; S.
(C-20), 155.13 (C-60). ESI MS m/z (%): 262.2 (61) [M+H], 284.2
(100) [M+Na]; HRMS ESI (C12H16O2N5): calculated: 262.12985,
found: 262.12988. For C12H15N5O2 (261.28): calculated: 55.16; C,
5.79; H, 26.80; N; found: 55.32; C, 5.85; H, 26.50; N.
4.14. tert-Butyl [(1R⁄,3R⁄,4R⁄)-3-hydroxybicyclo[2.2.1]hept-1-yl]
carbamate (39)
Boc2O (1.31 g, 6 mmol) was added to a solution of 7 (655 mg,
4 mmol) and DIPEA (2.09 mL, 12 mmol) in DCM (25 mL), and the
reaction mixture was stirred at rt overnight. DCM was evaporated,
and the resulting slurry was dissolved in ethyl acetate and washed
with water (2 ꢁ 50 mL). The organic layer was dried over sodium
sulfate, evaporated, and the crude product was purified by flash
chromatography (30–50% ethyl acetate in hexane) and crystallized
from hexane to afford 39 (807 mg, 89%) as white crystals.
4.13. Acid-catalyzed hydrolysis of 2-amino-6-chloropurine
substrates to guanine derivatives (compounds 17, 24, and 31)
A solution of a 2-amino-6-chloropurine derivative (0.5 mmol)
in a TFA–water mixture (2:1, 6 mL) was stirred at rt overnight.
The volatiles were evaporated and the crude product was codis-
tilled with ethanol (3 ꢁ 10 mL), NH4OH (10 mL) and ethanol
(2 ꢁ 10 mL). The poorly soluble product was shortly boiled in a
water–methanol mixture (1:1), collected on a filter and thoroughly
washed with water, ethanol and diethylether.
1H NMR (500 MHz, DMSO): d 1.12 (m, 1H, H-5endo), 1.34–1.40
(m, 2H, H-6endo, H-7b), 1.40 (s, 9H, CH3), 1.47 (dm, 1H, Jgem = 12.5,
H-2exo), 1.52–1.61 (m, 2H, H-5exo, H-6exo), 1.84 (dm, 1H,
Jgem = 9.1, H-7a), 1.89–1.92 (m, 2H, H-4, H-2endo), 3.66 (dm, 1H,
J3,2en = 7.0, H-3), 6.46 (br s, 1H, NH). 13C NMR (125.8 MHz, DMSO):
d 24.07 (C-5), 28.09 (CH3), 31.99 (C-6), 37.39 (C-7), 42.17 (C-4),
45.80 (C-2), 59.99 (C-1), 72.77 (C-3), 77.03 (CH3), 154.51 (COO).
ESI MS m/z (%): 250.1 (100) [M+Na]; HRMS ESI (C12H21O3NNa):
4.13.1. 2-Amino-9-[(1R⁄,3R⁄,4R⁄)-3-hydroxybicyclo[2.2.1]
hept-1-yl]-1,9-dihydro-6H-purin-6-one (17)
From 14 (100 mg, 0.36 mmol); yield: 52 mg, 56%, pale-orange
powder (mp >360 °C). 1H NMR (500 MHz, DMSO): d 1.25 (m, 1H,
H-5endo), 1.56 (dm, 1H, Jgem = 12.6, H-2exo), 1.67–1.77 (m, 2H,
H-6exo, H-5exo), 1.89 (m, 1H, H-6endo), 1.97 (dm, 1H, Jgem = 9.2,
H-7a), 2.11 (m, 1H, H-4); 2.27 (dm, 1H, Jgem = 9.2, H-7b), 2.48
(ddd, 1H, Jgem = 12.5, J2en,3 = 6.9, J2en,7a = 2.3, H-2endo), 3.79 (m,
1H, H-3), 4.82 (m, 1H, OH), 6.36 (br s, 2H, NH2), 7.66 (s, 1H,
H-80), 10.10 (br s, 1H, H-10). 13C NMR (125.8 MHz, DMSO): d
24.24 (C-5), 32.64 (C-6), 37.50 (C-7), 42.62 (C-4), 46.04 (C-2),
64.08 (C-1), 72.76 (C-3), 117.83 (C-50), 136.25 (C-80), 151.88
(C-40), 152.94 (C-20), 157.08 (C-60). ESI MS m/z (%): 262.2 (52)
[M+H], 284.2 (100) [M+Na]; HRMS ESI (C12H16O2N5): calculated:
calculated: 250.14136; found: 250.14125. For
C12H21NO3
(227.30): calculated: 63.41; C, 9.31; H, 6.16; N; found: 63.43; C,
9.33; H, 6.23; N.
4.15. The method of secondary-hydroxyl-group alkylation with
diisopropyl tosylmethanphosphonate (compounds 33, 35, 37
and 40)
A solution of diisopropyl tosylmethanphosphonate (1.5 mmol)
in DMF (5 mL) was added to a solution of a substrate (1 mmol)
and (t-BuO)2Mg (1.5 mmol) in dry DMF (25 mL), and the mixture
was heated to 60 °C for 3 d. The volatiles were evaporated and
the crude product was adsorbed on silica and purified by flash
chromatography to afford phosphonate diisopropyl ester.
262.12985, found: 262.12983. For
C12H15N5O2 (261.28):
calculated: 55.16; C, 5.79; H, 26.80; N; found: 55.09; C, 5.77; H,
26.71; N.
4.13.2. 2-Amino-9-[(1R⁄,4R⁄)-3-oxobicyclo[2.2.1]hept-1-yl]-
1,9-dihydro-6H-purin-6-one (24)
4.15.1. Diisopropyl [({4-[6-amino-9H-purin-9-yl]bicyclo[2.2.1]
hept-2-yl}oxy)methyl]phosphonate (33)
From 23 (150 mg, 0.54 mmol); yield: 120 mg, 86%, brown pow-
der (mp >360 °C (decomp)). 1H NMR (500 MHz, DMSO): d 1.50 (m,
1H, H-5endo), 2.05 (m, 1H, H-5exo), 2.13–2.22 (m, 2H, H-6), 2.37
(ddd, 1H, Jgem = 9.8, J7b,3en = 4.0, J7b,1 = 1.4, H-7b), 2.46 (dm, 1H,
Jgem = 9.8, H-7a), 2.68 (m, 1H, H-4), 2.69 (dd, 1H, Jgem = 17.2,
J2en,7b = 4.0, H-2endo), 2.77 (dm, 1H, Jgem = 17.2, H-2exo), 6.39 (br
s, 2H, NH2), 7.74 (s, 1H, H-80), 10.61 (br s, 1H, NH). 13C NMR
(125.8 MHz, DMSO): d 23.37 (C-5), 31.55 (C-6), 39.8 (C-7), 48.48
(C-2), 49.00 (C-4), 62.54 (C-1), 117.71 (C-50), 135.95 (C-80),
151.76 (C-40), 153.16 (C-20), 156.99 (C-60), 211.90 (C-3). ESI MS
m/z (%): 260.2 (40) [M+H], 282.2 (100) [M+Na]; HRMS ESI
(C12H13O2N5Na): calculated: 282.09615; found: 282.09613. For
From 10 (231 mg, 0.94 mmol); mobile phase: 3–10% methanol
in ethyl acetate; yield: 350 mg, 89%, clear oil. 1H NMR (500 MHz,
DMSO): d 1.24 (m, 12H, CH3-i-Pr); 1.30 (m, 1H, H-6endo), 1.81
(m, 1H, H-6exo), 1.84 (m, 1H, H-3exo), 1.91 (m, 1H, H-5exo), 1.97
(m, 1H, H-5endo); 2.03 (dm, 1H, Jgem = 9.4, H-7b), 2.28 (dm, 1H,
Jgem = 9.4, H-7a), 2.5 (m, 2H, H-1, H-3endo), 3.68–3.79 (m, 3H,
CH2P, H-2), 4.60 (m, 2H, CH-i-Pr), 7.18 (s, 2H, NH2), 8.11 (s, 1H,
H-20), 8.14 (s, 1H, H-80). 13C NMR (125.8 MHz, DMSO): d 23.87
(C-6), 23.89–24.06 (m, CH3-i-Pr), 32.61 (C-5), 37.83 (C-7), 38.61
(C-1), 43.11 (C-3), 62.54 (d, JC-P = 165.7, CH2-P), 64.15 (C-4), 70.35
(d, JC-P = 6.2, CH-i-Pr), 83.40 (d, J2-P = 13.1, C-2), 119.79 (C-50),
139.70 (C-80), 150.19 (C-40), 152.26 (C-20), 156.30 (C-60). ESI MS
m/z (%): 424.3 (100) [M+H], 446.3 (95) [M+Na]; HRMS ESI
(C19H30O4N5NaP): calculated: 446.19276; found: 446.19254. For
C12H13N5O2. 0.5 H2O (268.27): calculated: 53.72; C, 5.26; H,
26.11; N; found: 53.65; C, 5.19; H, 25.96; N.
4.13.3. 2-Amino-9-[(1R⁄,3R⁄,4R⁄)-3-hydroxybicyclo[2.2.1]hept-
1-yl]-1,9-dihydro-6H-purin-6-one (31)
C19H30N5O4P (423.45): calculated: 53.89; C, 7.14; H, 16.54; N;
7.31; P; found: 53.94; C, 7.23; H, 16.15; N, 7.38; N.
From 30 (100 mg, 0.36 mmol); yield: 60 mg, 64%, pale-orange
powder (mp >360 °C (decomp)). 1H NMR (500 MHz, DMSO): d
1.56 (m, 1H, H-6exo), 1.70 (dm, 1H, Jgem = 12.5, H-3endo), 1.82
(m, 1H, H-5exo), 2.02–2.18 (m, 4H, H-5endo, H-6endo, H-7), 2.15
(m, 1H, H-3exo), 2.19 (m, 1H, H-1), 4.21 (m, 1H, H-2), 4.88 (d,
1H, JOH-2 = 3.7, OH), 6.30 (br s, 2H, NH2), 7.60 (s, 1H, H-80), 10.52
(br s, 1H, H-10). 13C NMR (125.8 MHz, DMSO): d 24.71 (C-6),
33.80 (C-5), 40.05 (C-7), 40.88 (C-1), 42.80 (C-3), 66.48 (C-4),
69.91 (C-2), 117.45 (C-50), 133.01 (C-80), 152.44 (C-40), 153.88
4.15.2. Diisopropyl [({4-[6-(cyclopropylamino)-9H-purin-9-yl]
bicyclo[2.2.1]hept-2-yl}oxy)methyl]phosphonate (35)
From 11 (280 mg, 0.8 mmol); the mobile phase: 1–3% methanol
in ethyl acetate; yield: 70 mg, 25%, clear oil.
1H NMR (500 MHz, DMSO): d 0.66 and 0.92 (m, 4H, CH2-cyclop),
1.32–1.34 (m, 12H, CH3-iPr), 1.90–1.98 (m, 4H, H-3exo, H-5a, H-6),
2.09–2.16 (m, 2H, H-5b, H-7a), 2.40 (dm, 1H, Jgem = 9.2, H-7b), 2.57
(m, 1H, H-1), 2.69 (ddd, 1H, Jgem = 12.8, J3en-2 = 6.9, J3en-7a = 2.6,

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M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
H-3endo), 3.02 (br s, 1H, CH-cyclop), 3.69 (dd, 1H, Jgem = 13.4
JH-C-P = 9.5) and 3.74 (dd, 1H, Jgem = 13.4, JH-C-P = 9.4, CH2P), 3.79
(dm, 1H, J2-3en = 7.0, H-2), 4.71–4.80 (m, 2H, CH-iPr), 6.04 (br s,
1H, NH), 7.76 (s, 1H, H-80), 8.46 (s, 1H, H-20). 13C NMR
(125.8 MHz, DMSO): d 7.31 (CH2-cyclop), 23.98–24.09 (CH3-iPr),
24.20 (C-6), 33.02 (C-5), 38.16 (C-7), 38.90 (C-1), 43.35 (C-3),
63.26 (d, JC-P = 169.8, CH2P), 64.49 (C-4), 71.07 (d, JC-O-P = 6.7, CH-
iPr), 83.90 (d, J3-P = 12.7, C-2), 120.70 (C-50), 138.35 (C-80), 149.90
(C-40), 152.64 (C-20), 155.74 (C-60). HRMS ESI (C22H35N5O4P): calcu-
lated: 464.2421; found: 464.2420.
30–80% methanol in water, C18 column; yield: 107 mg, 60%, clear
solid or white lyophilizate. 1H NMR (500 MHz, D2O): d 1.34 (m, 1H,
H-5endo), 1.57–1.66 (m, 2H, H-2exo, H-6exo), 1.82–1.91 (m, 2H,
0
H-5exo, H-7b), 1.90 (d, 3H, JCH3,6 = 1.1, CH3), 2.13–2.24 (m,
2H, H-6endo, H-7a), 2.71 (ddd, 1H, Jgem = 13.0, J2en,3 = 6.9,
J2en,7b = 2.4, H-2endo), 3.68 (dd, 1H, Jgem = 13.7, JH,C,P = 9.3,
OCH2Pb), 3.73 (dd, 1H, Jgem = 13.7, JH,C,P = 9.1, OCH2Pa), 3.82 (m,
1H, H-3), 4.33 (d, 2H, JH,C,P = 12.4, NCH2Pb), 7.61 (br s, 1H, H-60).
13C NMR (125.8 MHz, D2O): d 12.72 (CH3), 24.36 (C-5), 31.59
(C-6), 38.69 (C-7), 39.02 (d, JC-P = 149.4, NCH2-P), 42.02 (C-2),
63.63 (d, JC-P = 159.8, OCH2-P), 71.44 (C-1), 84.79 (d, J2-P = 11.6,
C-3), 108.96 (C-50), 140.35 (C-60), 152.18 (C-20), 165.68 (C-40).
NegESI MS m/z (%): 423.1 (100) [MꢀH]; HRMS negESI
(C14H21O9N2P2) calculated: 423.07278; found: 423.07269. For
4.15.3. Diisopropyl [({4-[2-amino-6-(cyclopropylamino)-9H-
purin-9-yl]bicyclo[2.2.1]hept-2-yl}oxy)methyl] phosphonate (37)
From 16 (250 mg, 0.83 mmol); mobile phase: 1–2% methanol in
ethyl acetate; yield: 320 mg, 81%, clear oil. 1H NMR (500 MHz,
DMSO): d 0.57 and 0.65 (m, 4H, CH2-cyclop), 1.22–1.26 (m, 12H,
CH3-i-Pr), 1.26 (m, 1H, H-6endo), 1.71 (dm, 1H, Jgem = 12.8,
H-3exo), 1.75–1.85 (m, 2H, H-5exo, H-6exo), 1.93 (m, 1H,
H-5endo), 2.07 (dm, 1H, Jgem = 9.4, H-7b), 2.12 (dm, 1H, Jgem = 9.4,
H-7a), 2.45 (m, 1H, H-1), 2.54 (ddd, 1H, Jgem = 12.8, J3en,2 = 6.8,
J3en,7 = 2.2, H-3endo), 3.01 (br s, 1H, CH-cyclop), 3.69 (dd, 1H,
Jgem = 13.7, JH,C,P = 9.2, CH2Pa), 3.72 (m, 1H, H-2), 3.75 (dd, 1H,
Jgem = 13.7, JH,C,P = 8.9, CH2Pb), 4.55–4.64 (m, 2H, CH-i-Pr), 5.69
(br s, 2H, NH2), 7.25 (m, 1H, NH), 7.69 (s, 1H, H-80). 13C NMR
C14H22N2O9P2 (424.28): calculated: 39.63; C, 5.23; H, 6.60; N;
14.60; P; found: 39.81; C, 5.21; H, 6.40; N, 14.52; P.
4.16.2. [({4-[6-Amino-9H-purin-9-yl]bicyclo[2.2.1]hept-2-
yl}oxy) methyl]phosphonic acid (34)
From 33 (250 mg, 0.59 mmol); yield: 190 mg, 95%, poorly-solu-
ble white powder (mp = 289–290 °C). 1H NMR (500 MHz, D2O): d
1.42 (m, 1H, H-6endo), 1.82 (m, 1H, H-5exo), 1.87–1.94 (m, 2H,
H-3exo, H-6exo), 1.97–2.03 (m, 2H, H-5endo, H-7b), 2.38 (dm,
1H, Jgem = 9.1, H-7a), 2.55 (m, 1H, H-3endo), 2.60 (bd, 1H,
J1,6ex = 5.0, H-1), 3.43 (dd, 1H, Jgem = 12.5, JH,C,P = 9.3, CH2Pb), 3.49
(dd, 1H, Jgem = 12.5, JH,C,P = 9.0, CH2Pa), 3.87 (m, 1H, H-2),
8.12–8.14 (m, 2H, H-20, H-80). 13C NMR (125.8 MHz, D2O):
d 24.52 (C-6), 32.90 (C-5), 38.51 (C-7), 39.33 (C-1), 43.30 (C-3),
65.44 (C-4), 66.65 (d, JC-P = 151.0, CH2-P), 83.98 (d, J2-P = 11.0,
C-2), 119.87 (C-50), 141.95 (C-80), 149.86 (C-40), 152.38 (C-20),
156.05 (C-60). NegESI MS m/z (%): 338.1 (100) [MꢀH]; HRMS
negESI (C13H17O4N5P): calculated: 338.10236; found: 338.10268.
For C13H18N5O4P (339.29): calculated: 46.02; C, 5.35; H, 20.64; N;
9.13; P; found: 46.37; C, 5.21; H, 20.29; N, 9.02; P.
(125.8 MHz, DMSO):
d 6.58 (CH2-cyclop), 23.88–24.04 (m,
CH3-i-Pr), 23.99 (C-6), 32.43 (C-5), 37.75 (C-7), 38.60 (C-1), 43.07
(C-3), 62.51 (d, JC-P = 165.8, CH2-P), 63.62 (C-4), 70.28 (m,
CH-i-Pr), 83.37 (d, J2-P = 13.0, C-2), 114.52 (C-50), 135.91 (C-80),
152.0 (C-40), 156.11 (C-60), 159.87 (C-20). NegESI MS m/z (%):
423.1 (100) [MꢀH]; HRMS negESI (C14H21O9N2P2): calculated:
423.07278; found: 423.07269. For C22H35N6O4P (478.52): calcu-
lated: 55.22; C, 7.37; H, 17.56; N; 6.47; P; found: 55.28; C, 7.47;
H, 17.30; N, 6.78; P.
4.15.4. Diisopropyl [({(1R⁄,3R⁄,4R⁄)-4-[(tert-butoxycarbonyl)
amino]bicyclo[2.2.1]hept-2-yl}oxy)methyl]phosphonate (40)
From 39 (770 mg, 3.39 mmol); mobile phase: 60–100% ethyl ace-
tate in hexane; yield: 800 mg, 58%, clear oil. 1H NMR (500 MHz,
DMSO): d 1.09 (m, 1H, H-6endo), 1.22–1.25 (m, 12H, CH3CH), 1.37
(s, 9H, CH3C), 1.33–1.40 (m, 2H, H-5endo, H-7b), 1.50–1.62 (m, 3H,
H-3exo, H-6exo, H-5exo), 1.67 (m, 1H, H-7a), 1.88 (m, 1H, H-3endo),
2.20 (m, 1H, H-1), 3.51 (m, 1H, H-2), 3.60 (dd, 1H, Jgem = 13.7,
JH,C,P = 9.3, CH2Pb), 3.68 (dd, 1H, Jgem = 13.7, JH,C,P = 9.0, CH2Pa),
4.54–4.63 (m, 2H, CH3CH), 7.03 and 7.32 (br s, 1H, NH). 13C NMR
(125.8 MHz, DMSO): d 23.87–24.05 (m, CH3CH), 24.06 (C-6), 28.46
(CH3C), 32.07 (C-5), 37.83 (C-7), 38.13 (C-1), 42.74 (C-3), 60.09
(C-4), 62.48 (d, JC-P = 166.0, CH2-P), 70.21–70.29 (m, CH3CH), 77.48
(CH3C), 83.89 (d, J2-P = 13.3, C-2), 154.80 and 154.97 (COO). ESI MS
m/z (%): 406.3 (17) [M+H], 428.3 (100) [M+Na]; HRMS ESI
(C19H36O6NNaP): calculated: 428.21725; found: 428.21728.
4.16.3. [({4-[6-(Cyclopropylamino)-9H-purin-9-yl]bicyclo[2.2.1]
hept-2-yl}oxy)methyl]phosphonic acid (36)
From 35 (70 mg, 0.15 mmol); yield: 53 mg, 92%, white solid. 1H
NMR (500 MHz, D2O): d 0.90 and 1.12 (m, 4H, CH2-cyclop), 1.62 (m,
1H, H-6endo), 1.94–2.01 (m, 3H, H-3exo, H-5endo, H-6exo),
2.11–2.16 (m, 2H, H-5exo, H-7a), 2.47 (dm, 1H, Jgem = 9.6, H-7b),
2.65 (m, 1H, H-1), 2.68 (ddd, 1H, Jgem = 12.7, J3en-2 = 6.8,
J3en-7a = 2.5, H-3endo), 2.91 (br s, 1H, CH-cyclop), 3.73 (dd, 1H,
Jgem = 13.7, JH-C-P = 9.4, CH2Pa), 3.80 (dd, 1H, Jgem = 13.7,
JH-C-P = 9.1, CH2Pb), 3.94 (dm, 1H, J2-3en = 6.8, H-2), 8.36 (s, 1H,
H-80), 8.43 (s, 1H, H-20). 13C NMR (125.8 MHz, D2O): d 7.31
(CH2-cyclop), 23.33 (CH-cyclop), 24.25 (C-4), 33.15 (C-5), 38.43
(C-7), 39.45 (C-1), 43.41 (C-3), 63.93 (d, JC-P = 159.9, (CH2P), 66.14
(C-4), 71.07 (d, JC-O-P = 6.7, CH-iPr), 83.90 (d, J3-P = 12.7, C-3),
120.70 (C-50), 138.35 (C-80), 149.90 (C-40), 152.64 (C-20), 155.74
(C-60). HRMS ESI (C16H22N5O4P): calculated: 380.1482; found:
380.1482.
4.16. The method of hydrolysis of phosphonate diesters
(compounds 32, 34, 36 and 38)
4.16.4. [({4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-
TMSBr (0.7 mL) was added to a solution of diisopropyl phospho-
nate (0.5 mmol) in dry DCM (10 mL) and the reaction mixture was
stirred at rt for 24 h. The volatiles were evaporated and the crude
product was codistilled with dry ethanol (3 ꢁ 15 mL) to afford free
phosphonate as a clear solid or white lyophilizate. Further purifica-
tion was possible on a short C18 column.
yl]bicyclo [2.2.1]hept-2-yl}oxy)methyl]phosphonic acid (38)
From 37 (220 mg, 0.46 mmol); the mobile phase: (30–80%
methanol in water); yield: 164 mg, 90%, clear solid or white
lyophilizate. 1H NMR (500 MHz, D2O): d 0.83 and 1.05 (m, 4H,
CH2-cyclop), 1.36 (m, 1H, H-6endo), 1.79–1.98 (m, 5H, H-5,
H-3exo, H-6exo, H-7b), 2.30 (dm, 1H, Jgem = 9.4, H-7a), 2.54 (ddd,
1H, Jgem = 12.6, J3en,2 = 6.9, J3en,7b = 2.3, H-3endo), 2.56 (bd, 1H,
J1,6ex = 4.8, H-1), 2.84 (br s, 1H, CH-cyclop), 3.59 (dd, 1H, Jgem = 13.3,
JH,C,P = 9.5, CH2Pb), 3.67 (dd, 1H, Jgem = 13.3, JH,C,P = 9.0, CH2Pa), 3.83
(m, 1H, H-2), 7.92 (s, 1H, H-80). 13C NMR (125.8 MHz, D2O): d 7.53
(CH2-cyclop), 24.14 (C-6), 32.85 (C-5), 38.13 (C-7), 39.19 (C-1),
43.02 (C-3), 64.81 (d, JC-P = 156.9, CH2-P), 65.43 (C-4), 84.36
4.16.1. ({5-methyl-2,6-Dioxo-3-[(1R⁄,3R⁄,4R⁄)-3-(phosphono-
methoxy)bicyclo[2.2.1]hept-1-yl]-3,6-dihydropyrimidin-1(2H)-
yl}methyl)phosphonic acid (32)
From 8 (100 mg, 0.42 mmol); the mobile phase: 1–2% methanol
in ethyl acetate; after hydrolysis of ester groups: the mobile phase:

M. Dejmek et al. / Bioorg. Med. Chem. 22 (2014) 2974–2983
2983
(d, J2-P = 11.9, C-2), 112.50 (C-50), 141.51 (C-80), 151.4 (C-40), 150.63
and 152.47 (C-20, C-60). NegESI MS m/z (%): 393.1 (100) [MꢀH];
HRMS negESI (C16H22O4N6P): calculated: 393.14456; found:
393.14462. For C16H23N6O4P (394.37): calculated: 48.73; C, 5.88;
H, 21.31; N; 7.85; P; found: 48.91; C, 5.76; H, 21.33; N, 7.72; P.
the compound concentration required to reduce virus-induced
cytopathogenicity or viral plaque formation by 50%.
4.19. Anti-HIV activity assays
The inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cyto-
pathicity in CEM cell cultures was measured in microtiter 96-well
plates containing ꢂ3 ꢁ 105 CEM cells/mL infected with 100 CCID50
of HIV per milliliter and containing appropriate dilutions of the test
compounds. After 4ꢀ5 d of incubation at 37 °C in a CO2-controlled
humidified atmosphere, CEM giant (syncytium) cell formation was
examined microscopically. The EC50 (50% effective concentration)
was defined as the compound concentration required to inhibit
HIV-induced giant-cell formation by 50%.
4.17. ({[(1R⁄,3R⁄,4R⁄)-4-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)bicyclo[2.2.1]hept-2-yl]oxy}methyl)
phosphonic acid (42)
TFA (2 mL) was added to the solution 40 (800 mg) in DCM
(10 mL) and the reaction mixture was stirred at rt for 2 h. The vol-
atiles were evaporated, and the product was codistilled with dry
ethanol (3 ꢁ 15 mL) and purified on Dowex 50 (H+) to afford 41
(320 mg, 56%) as brownish oil, which was directly used in the next
reaction. Ethyl [(2E)-3-ethoxy-2-methylprop-2-enoyl]carbamate
Acknowledgments
(203 mg, 1 mmol) was added to
a solution of 41 (280 mg,
0.92 mmol) in dioxane (15 mL) and the reaction mixture was
heated at 100 °C for 3 h. Dowex 50 (H+, 5 g) was then added and
the reaction mixture was heated to 100 °C overnight. The Dowex
resin was filtered off, the crude product was adsorbed on C18 silica
and purified by flash chromatography on the reverse-phase
column (30–80% methanol in water) 42 (130 mg, 43%) as white
lyophilizate. 1H NMR (500 MHz, D2O): d 1.21 (m, 1H, H-6endo),
1.41–1.51 (m, 2H, H-3exo, H-5exo), 1.77 (d, 3H, CH3), 1.69–1.80
(m, 2H, H-6exo, H-7b), 2.02–2.11 (m, 2H, H-5exo, H-7a), 2.34 (d,
1H, J1,6ex = 5.2, H-1), 2.59 (ddd, 1H, Jgem = 12.9, J3en,2 = 2.1,
J3en,7a = 2.1, H-3endo), 3.44 (dd, 1H, Jgem = 13.2, JH,C,P = 9.6, CH2Pb),
3.49 (dd, 1H, Jgem = 13.2, JH,C,P = 9.7, CH2Pa), 3.63 (d, 1H,
This study was supported by the Ministry of the Interior of the
Czech Republic (VG20102015046) and Gilead Sciences, Inc. (Foster
City, CA, USA). Subvention for development of research organiza-
tion (RVO: 61388963) is also acknowledged. The biological exper-
iments were carried out with the financial support of the KU
Leuven (GOA 10/14).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.004.
0
J2,3en = 6.6, H-2), 4.67 (br s, 2H, POH), 7.50 (d, 1H, J6 ,CH3 = 1.1,
H-60), 11.11 (s, 1H, NH). 13C NMR (125.8 MHz, D2O): d 12.18
(CH3), 23.99 (C-6), 31.47 (C-5), 37.46 (C-1), 38.31 (C-7), 42.19
(C-3), 63.96 (d, JC-P = 162.5, CH2-P), 68.78 (C-4), 83.06 (d,
J2-P = 12.4, C-2), 107.93 (C-50), 139.92 (C-60), 151.09 (C-20), 164.31
(C-40). NegESI MS m/z (%): 329.2 (100) [MꢀH]; HRMS NegESI
(C13H18O6N2P): calculated: 329.08970; found: 329.08997.
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The compounds were evaluated against the following viruses:
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