Synthesis of novel purine nucleosides towards a selective inhibition of human butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2009.05.057

The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic chemistry groups around the world as these molecules have been shown to treat the late symptoms of Alzheimer's disease as well as to act as neuroprotecting

Full text of DOI:10.1016/j.bmc.2009.05.057

Bioorganic & Medicinal Chemistry 17 (2009) 5106–5116
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel purine nucleosides towards a selective inhibition of human
butyrylcholinesterase
Filipa Marcelo a,c, Filipa V. M. Silva a,b, Margarida Goulart a,b, Jorge Justino b, Pierre Sinaÿ c,
Yves Blériot c, Amélia P. Rauter a,
*
a Carbohydrate Chemistry Group, CQB-FCUL, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande,
Ed. C8, 5° Piso, Campo Grande, 1749-016 Lisboa, Portugal
b Escola Superior Agrária de Santarém, Apartado 310, 2001-904 Santarém, Portugal
c UMR 7201, Institut Parisien de Chimie Moléculaire, Equipe Glycochimie, UPMC-Paris 6, 4 Place de Jussieu, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic
chemistry groups around the world as these molecules have been shown to treat the late symptoms of
Alzheimer’s disease as well as to act as neuroprotecting agents. In this work, we disclose the synthesis
of novel 2-acetamidopurine nucleosides and, for the first time, regioselective N7-glycosylation with 2-
acetamido-6-chloropurine, promoted by trimethylsilyl triflate, was accomplished by tuning the reaction
conditions (acetonitrile as solvent, 65 °C, 5 h) starting from 1-acetoxy bicyclic glycosyl donors, or by
direct coupling of a methyl glucopyranoside with the nucleobase to obtain only N7 nucleosides in reason-
able yield (55–60%). The nucleosides as well as their sugar precursors were screened for acetylcholines-
terase (AChE) and butyrylcholinesterase (BChE) inhibition. While none of the compounds tested inhibited
AChE, remarkably, some of the N7 nucleosides and sugar bicyclic derivatives showed potent inhibition
towards BChE. Nanomolar inhibition was obtained for one compound competing well with rivastigmine,
a drug currently in use for the treatment of Alzheimer’s disease. Experimental results showed that the
presence of benzyl groups on the carbohydrate scaffold and the N7-linked purine nucleobase were nec-
essary for strong BChE inactivation. A preliminary evaluation of the acute cytotoxicity of the elongated
bicyclic sugar precursors and nucleosides was performed indicating low values, in the same order of mag-
nitude as those of rivastigmine.
Received 1 January 2009
Revised 15 May 2009
Accepted 23 May 2009
Available online 29 May 2009
Keywords:
Purine nucleosides
Regioselective N7-glycosylation
BChE inhibition
Alzheimer disease
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
icit of cerebral ACh. However, in advanced AD, AChE levels in the
brain have already decreased, while BChE activity is still high, sug-
There are two major forms of cholinesterases, acetylcholinester-
ase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8)
in mammalian tissues. Both AChE and BChE belong to the group of
serine hydrolases and are responsible for the breakdown of the
neurotransmitter acetylcholine (ACh) and of butyrylcholine
(BCh), respectively.1 In Alzheimer’s disease (AD) the loss of cholin-
ergic neurotransmission in the brain is accompanied by a reduced
concentration of ACh and contributes to the salient cognitive and
behavioral disturbances characteristic of AD.2–4 This is the most
common type of dementia in western societies, which has been
causing profound economic and social impact as the aging popula-
tion increases.5 The cholinergic hypothesis6 represents one of the
most promising approaches involving the design of new agents
for the treatment of AD. This strategy is based on the development
of drugs with an AChE inhibition profile in order to rectify the def-
gesting that ACh hydrolysis may occur to a greater extent via BChE
catalysis.7 In fact, it has been reported that the specific inhibition of
BChE is important in raising acetylcholine levels and improving
cognition.8,9
Cholinesterase inhibitors such as tacrine,10 rivastigmine,11
donepezil12,13 and galanthamine,14 currently in use to treat AD, in-
hibit both AChE and BChE,15 and it is difficult to determine
whether the positive effects observed result from inhibition of
AChE or BChE enzymes or both. For this reason, it is important to
design selective, potent and well-tolerated inhibitors of each cho-
linesterase in order to determine which enzyme needs to be tar-
geted for maximum effect in treating AD.16
2-Aminopurine nucleosides are rarely found in Nature. How-
ever such N9 nucleosides are key structural features of Amypuri-
mycin and Miharamycins A and B, three natural products which
are potent inhibitors of Pyricularia oryzae, a fungus responsible
for the rice blast disease. We have previously reported the first to-
tal synthesis of the core of Miharamycin B, which includes the syn-
thesis of several structural fragments, such as N9 nucleosides
* Corresponding author. Tel.: +351 21 7500952; fax: +351 21 7500088.
E-mail addresses: jorge.justino@ipsantarem.pt (J. Justino), aprauter@fc.ul.pt,
aprauter@gmail.com (A.P. Rauter).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.057

F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
5107
incorporating a complex bicyclic carbohydrate skeleton.17,18 Here-
in we report the synthesis of novel 2-acetamidopurine N7 nucleo-
sides, in which this unusual bicyclic sugar moiety is present
(compounds type A, Fig. 1). Our interest in the development of
selective cholinesterase inhibitors prompted us to evaluate these
molecules, as well as their precursors, as AChE and BChE inhibitors.
In addition, a preliminary screening of the acute cytotoxicity of this
family of compounds completed the biological studies.
Sinaÿ.19 Benzylidene hydrolysis cleanly afforded the unprotected
bicyclic scaffold 2, which was peracetylated and perbenzoylated
to give compounds 3 and 4 in good yield, respectively (Scheme 1).
As part of an ongoing program to exploit the total synthesis of
the natural Miharamycins antibiotics,17 we devised a non-stereo-
selective chain extension at C-6 of the corresponding primary alco-
hol 5. Its Swern oxidation proceeded efficiently to give the crude
aldehyde, which was used directly without further purification
(Scheme 2). In order to obtain both diastereoisomers at C-60, viny-
lation of the aldehyde in the absence of chelating species proved to
be the method of choice to obtain both allylic alcohols 6a and 6b.17
Their conversion into the corresponding azido esters was then
studied. Since the azido group is stable in the presence of oxidants,
it could be either introduced early in the synthesis, before the oxi-
dative cleavage of the vinyl group, or after the introduction of the
carboxylic acid function. Preliminary studies showed that azide
displacement of the triflate derived from the allylic alcohol 6a
mainly afforded the regioisomeric azide resulting from an allylic
rearrangement. Czernecki’s group also reported an allylic rear-
rangement when he tried to introduce the azide via Mitsunobu
reaction on a hexodialdo-1,5-pyranose.20 These results prompted
us to synthesize the hydroxy esters first.
2. Results and discussion
A total of fourteen compounds including nucleosides and their
bicyclic sugar precursors were synthesized and evaluated for their
ability to inhibit human serum BChE and bovine erythrocyte AChE
(BoAChE). The free bicyclic sugar, its acyl and benzyl protected
derivatives, the sugar azido esters and the two regioisomeric
nucleosides resulting from N-glycosylation of the purine base were
screened. A strong and selective inhibition for BChE was observed
for some of the products, and compared to rivastigmine. The N7
nucleosides were the most promising compounds and for that pur-
pose, stereo- and regioselective coupling conditions were devised
in order to obtain only the N7 regioisomers. A preliminary toxicity
screening of closely related elongated sugar precursors and purine
nucleosides was also accomplished.
Ozonolysis of the allylic alcohols 6a and 6b followed by sodium
chlorite oxidation in tBuOH/H2O/2-methylbut-2-ene (2:2:1) affor-
ded the corresponding carboxylic acids, which were not isolated.
Esterification with benzyl bromide in the presence of potassium
hydrogenocarbonate gave the corresponding benzyl esters 7a and
7b (Scheme 2) which were converted to the corresponding triflates.
Azide displacement with sodium azide in DMF at room tempera-
ture afforded the inverted azido esters in good yield (Scheme 2).
In order to obtain both N9 and N7 nucleosides 11 and 12 our
gained knowledge concerning N-glycosylation with glycosyl
donors such as 9 was exploited,17,18 and their coupling with the
persilylated 2-acetamido-6-chloropurine 10 was accomplished
using the conditions described in Scheme 3.
2.1. Chemistry
The synthesis of the bicyclic sugar moiety 1 was carried out
according to the strategy previously developed by Fairbanks and
Cl
N
R'
O
O
RO
N
N
RO
RO
N
NHAc
Since the N7 regioisomer 12 was the most promising compound
to inhibit the BChE (Table 2), regioselective coupling conditions
were developed to furnish only N7 nucleosides. In order to obtain
A
Figure 1. General structure of the new synthetic 2-acetamidopurine nucleosides.
O
HO
HO
RO
Ph
O
O
O
O
O
a)
O
RO
b) or c)
HO
HO
HO
HO
RO
RO
O
OMe
OMe
OMe
1
2
3
R = Ac
R = Bz 4
Scheme 1. Synthesis of the bicyclic sugar moieties 2, 3 and 4. Reagents and conditions: (a) AcOH (80% aq), 50 °C, quant; (b) Ac2O, pyridine, DMAP, rt, 92% for 3; (c) BzCl,
pyridine, DMAP, rt?80 °C, 72% for 4.
R
R
OH
HO
HO
O
O
O
O
O
O
a)
BnO
BnO
BnO
BnO
+
BnO
BnO
BnO
OMe
OMe
OMe
BnO
BnO
5
6a R = CH=CH2 (6S)
7a
6b R = CH=CH2 (6R)
7b R = COOBn (6S)
b)
b)
R = COOBn (6R)
COOBn
COOBn
N3
N3
(S)
(R)
O
O
c)
BnO
BnO
+
BnO
BnO
BnO
BnO
O
O
OMe
OMe
8a
8b
Scheme 2. Synthesis of the benzyl azido esters 8a and 8b. Reagents and conditions: (a) (COCl)2, DMSO, Et3N, ꢀ78 °C then CH2@CHMgBr, THF, ꢀ78 °C, 65% over two steps;17
(b) O3, DMS, CH2Cl2, ꢀ78 °C, then NaClO2, NaH2PO4.H2O, tBuOH/H2O/2-methylbut-2-ene (2:2:1), then BnBr, KHCO3, Bu4NI, DMF, 68% over three steps; (c) Tf2O, pyridine,
CH2Cl2, ꢀ78 °C, then NaN3, DMF 72% for 8a over two steps, 86% for 8b over two steps.

5108
F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
OAc
Cl
N
N
O
BnO
BnO
N
N
O
NHAc
BnO
OH
OAc
11
O
O
c)
O
a), b)
N9 regioisomer
+
HO
BnO
BnO
OAc
HO
HO
Cl
O
OMe
N
N
OAc
N
BnO
OTMS
Me
N
TMS
2
9
O
N
N
N
BnO
NHAc
N
BnO
10
N
O
Cl
BnO
12
N7 regioisomer
Scheme 3. Synthesis of the N9- and N7 nucleosides 11 and 12. Reagents and conditions: (a) NaH (60% w/w), BnBr, DMF, 85%; (b) 5% concd H2SO4 in AcOH, Ac2O, ꢀ20 °C? 0 °C,
48%; (c) TMSOTf, (CH2Cl)2, 85 °C, 25% for 11 and 37% for 12.
the suitable glycosyl donors, methyl glycosides 8a and 8b were
transformed into their more reactive 1-acetoxy counterparts 13a
and 13b (Scheme 4). Acetolysis of the tribenzylated 6S diastereo-
isomer 8a took place under the conditions previously optimized
to avoid the formation of ring contraction products,17,18 namely
in the presence of catalytic concentrated sulfuric acid 5% in acetic
acid, at low temperature, while acetolysis of its 6R epimer oc-
curred smoothly and in high yield under catalysis of concentrated
sulfuric acid, emphasizing the acidic sensitivity of the 6S isomer.
Regioselective synthesis of N7 nucleosides under kinetic control
(SnCl4 as Lewis acid, CH3CN as polar solvent and at low tempera-
ture) has been previously reported by Garner.21 When applying
these conditions, unsatisfactory yields were obtained for the b-
N7-nucleosides 14a and 14b (Scheme 4) which could be related
to the presence of the benzyl ethers, which are sensitive to the
strong acidic conditions22 required for the N-glycosylation. In or-
der to improve the yield various conditions were applied and it
was found that the use of the milder TMSOTf Lewis acid as pro-
moter at 65 °C furnished the b-N7-nucleosides 14a and 14b in sat-
isfactory yield. Considering the problematic acetolysis of the 6S
epimer, the direct coupling of the methyl glycoside 8a with the
persilylated base was successfully attempted using TMSOTf
(20 equiv) in CH3CN at 65 °C for 5 h to give the target N7 nucleo-
side in 60% yield. When these conditions were applied to the
methyl glycoside 8b the N7 nucleoside was also isolated as single
product in 55% yield.
2.2. Enzymatic studies
The inhibitory activity of the new synthetic compounds against
both AChE (bovine erythrocytes) and BChE (human serum) was
evaluated using Ellman’s spectrophotometric method23 to deter-
mine the rate of hydrolysis of acetylthiocholine and butyrylthioch-
oline, respectively, in the presence of the inhibitor. The activity of
the enzymes produces a yellow compound which was monitored
spectrophotometrically (k = 410 nm) along the reaction time. The
enzyme activity (%) and the enzyme inhibition (%) were calculated
from the rate of absorbance change with time (V =
as follows:
DAbs/Dt) data
Enzyme Inhibition ð%Þ ¼ 100 ꢀ Enzyme Activity ð%Þ
Enzyme Activity ð%Þ ¼ 100 ꢁ V=Vmax
Maximum rates (Vmax) are obtained when no inhibitor is used
while V is the rate obtained in the presence of the inhibitor.
The t-test (one-sided) was carried out in order to evaluate if the
average inhibition of the enzymes with compounds tested and the
positive control rivastigmine (drug available on the market), are
significantly higher than the average inhibition (0%) obtained in
the assay without any inhibitor. The t-test gives a probability be-
tween 0.00 and 1.00. When the probability 60.05 or 5%, this means
that the inhibition obtained with the compound at a certain
concentration is significant. The IC50 is an estimate value of the
COOBn
N3
COOBn
N3
O
O
BnO
BnO
BnO
a)
OAc
BnO
BnO
O
O
OMe
BnO
Cl
(6S) 13a
(6R) 13b
8a
(6S)
(6R)
N
N
N
OTMS
Me
8b
TMS
N
N
d)
b) or c)
10
COOBn
O
N3
N
N
N
BnO
NHAc
N
BnO
BnO
O
Cl
N7 regioisomer
14a
(6S)
(6R) 14b
Scheme 4. Synthesis of the (60S)- and (60R)-configurated N7 nucleosides 14a and 14b. Reagents and conditions: (a) 5% concd H2SO4 in AcOH, Ac2O, ꢀ20 °C?0 °C, 36% for 13a
and concd H2SO4, Ac2O, 0 °C, 63% for 13b; (b) SnCl4, CH3CN, rt 32% for 14a and 28% for 14b; (c) TMSOTf, CH3CN, 65 °C, 65% for 14a and 58% for 14b; (d) TMSOTf, CH3CN, 65 °C,
60% for 14a and 55% for 14b.

F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
5109
Table 1
compound concentration which inhibits 50% of the enzyme activ-
ity. IC50 values were determined for the active compounds and riv-
astigmine using non-linear regression analysis (Statistica 8.0 by
StatSoft, Inc., USA) toward a dose–response curve (enzyme inhibi-
tion vs substance concentration) and fitting an appropriate mathe-
matical model.
The BoAChE and human AChE (HuAChE) display identical
structure characteristics. Mendelson’s et al. have mapped onto the
three-dimensionalmodelof HuAChE, wherealldivergentaminoacids
between BoAChE and HuAChE were shown.24 The authors reported
that all the 34 divergent amino acids are located at the protein surface
andthesestructuraldifferencescouldonlyleadtofunctionalmanifes-
tations not related to the cholinergic activities. For this reason identi-
cal kinetic behavior ofBoAChEandHuAChEwith respect tohydrolysis
and binding with different inhibitors should be expected.24
None of the compounds tested showed a significant inhibition
of BoAChE. Noteworthy, inhibition of BChE (human serum) was de-
tected for most of the studied compounds (Table 1). The presence
of the benzyl groups seems to improve BChE inhibition if we com-
pare the results obtained for the free and acylated derivatives (2–4)
and for the benzylated derivative 5. The azido group also appears
to play a role for the inhibition of the BChE. Nevertheless the inhi-
bition results obtained do not allow to establish a structure–activ-
ity relationship regarding the configuration at C-6.
Regarding the BChE inhibitory profile of nucleosides 11 and 12,
only N7 regioisomer proved to be a good inhibitory agent (Table 2).
Nevertheless, the two diastereoisomers at C-60 14a and 14b
showed a remarkable BChE inhibition and especially compound
14b with R configuration at C-60 showed an IC50 of the same order
of magnitude as that of the control, rivastigmine.
Inhibition (%) of BChE for different concentrations of the compounds tested and IC50
values
Compound tested
Concentration BChE inhibition
BChE IC50 SEM
QiM)a
(
lg/mL)
(%)
HO
O
HO
HO
100
100
100
<5
—
O
OMe
HO
2
AcO
O
O
AcO
AcO
AcO
<5
—
OMe
OMe
3
BzO
BzO
BzO
BzO
O
O
7
—
4
HO
O
O
BnO
BnO
BnO
100.00
10.00
60***
<5
148.00 43.00
OMe
OMe
5
HO
(S)
O
O
Interestingly, experimental results showed a good and selective
inhibition of BChE vs AChE for the N7 nucleosides (12, 14a,b) and
some of the bicyclic sugar precursors (7a, 8a,b). These two en-
zymes have a significant homology and they mainly differ by the
volume of their active site gorge. In AChE the estimated volume
is relatively small (302 Å3), being lined with 14 bulky aromatic
amino acid residues, while that of BChE, with only 8 aryl residues,
is considerably larger (502 Å3).25,26 In addition AChE active site has
an elevated electronegative potential and an anionic peripheral site
while BuChE has a lower electronegative potential and a non-anio-
nic peripheral site. Since the experiments were run at pH 8, proton-
ation of the studied compounds is not expected. Hence, the volume
of the molecules’ active moiety may play an important role in the
observed selectivity. Alternatively, it can be hypothesized that the
binding site of the new ligands may be different from the catalytic
gorge. The bioactivity shown by the benzylated derivatives also
indicates that the binding site at BChE seems highly lipophilic, a
characteristic known for its catalytic site as reported by other
BnO
BnO
BnO
***
100.00
10.00
1.00
76
54***
4
31.00 2.90
6a
HO
(R)
O
O
BnO
100.00
10.00
73***
23***
BnO
BnO
50.00 4.60
OMe
6b
COOBn
HO
(R)
O
100.00
10.00
1.00
95***
56***
7
BnO
BnO
BnO
16.10 0.90
O
OMe
authors.27 Additionally,
p–p interaction has been reported to take
7a
place between the heterocyclic ring system of known inhibitors
and the aromatic amino acid residues F329 and Y332 of BChE.28,29
Thus, p–p interaction could explain the better inhibition found for
COOBn
the compounds containing the heterocyclic purine base. Neverthe-
less, it should be noted that the pattern of activity displayed by N7
and N9 regioisomers is undoubtedly divergent. Apparently, the
structural differences between these two regioisomers play an
important role in BChE profile inhibition.
HO
(S)
O
100.00
10.00
55***
8
133.00 27.00
BnO
BnO
BnO
O
OMe
7b
2.3. Preliminary acute toxicity screening
COOBn
(S)
N3
O
Considering the inhibition activity of some of the synthetic
compounds towards BChE, it became interesting to explore further
application in the treatment or palliation of Alzheimer’s disease
symptoms. The first concern in this matter would be the potential
toxicity of such compounds family, and therefore the sugar precur-
sors 7a, 8a, 8b and the readily available nucleosides 14a, 15a17 and
91***
64***
36***
15*
BnO
100.00
10.00
1.00
BnO
BnO
O
OMe
4.20 0.40
8a
0.10
(continued on next page)

5110
F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
Table 1 (continued)
Table 2
Inhibition (%) of BChE for different concentrations of the compounds tested and IC50
values
Compound tested
Concentration BChE inhibition
BChE IC50 SEM
QiM)a
(lg/mL)
(%)
Compound tested
Concentration
g/mL)
BChE
inhibition
(%)
BChE
COOBn
(R)
(
l
IC50 SEMa
N3
(lM)
O
O
100.00
10.00
1.00
98***
73***
14**
13.40 0.80
BnO
Cl
N
N
OAc
BnO
BnO
N
N
OMe
O
100.00
10.00
48***
11
—
BnO
NHAc
BnO
8b
O
BnO
11
Rivastigmineb
100.00
10.00
1.00
0.10
0.01
100***
100***
100***
76***
NI
0.17 0.01
OAc
N
NHAc
N
N
O
BnO
BnO
BnO
84***
85***
70***
20***
0.76 0.05
N
100.00
10.00
1.00
Cl
O
a
IC50 is an estimate of the compound concentration which inhibits 50% of the
enzyme activity; values are expressed as mean standard error of the mean (SEM).
12
0.10
b
Rivastigmine is a standard drug to treat AD patients and was used as positive
control. ***P < 0.001, **P < 0.01, *P < 0.05.
COOBn
N3
(
)
S
N
NHAc
N
N
15b18 (Table 3) were selected as probe compounds for a prelimin-
ary screening of acute in vitro toxicity. Cytotoxicity was assessed
by the quantification of viable and metabolically active continuous
culture human cells following a 24 h exposure to the drug by the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] cell proliferation assay.30,31 A similar toxicity profile was
exhibited by these probe compounds, namely 8a and 8b showed
the same behavior, suggesting that configuration at C-6 does not
influence the toxic effect. There was a subtle trend for a slightly
higher toxicity of the N7 nucleoside 14a, while the less toxic mol-
ecule appeared to be 7a, with a free OH-6. These data only give an
estimate of the potential toxicity of this family of compounds.
However the toxicity shown can still be considered low when com-
pared to the one exhibited by rivastigmine, a standard drug cur-
rently used in the treatment of patients suffering from
Alzheimer’s disease.
O
BnO
N
100.00
10.00
1.00
81***
34***
9
22.00 1.60
BnO
BnO
Cl
O
14a
COOBn
N3
N
N
Cl
NHAc
(R)
N
N
O
BnO
100
10
1
0.1
0.01
91***
79***
77***
63***
10
0.14 0.01
BnO
BnO
O
14b
Rivastigmineb
100
10
1
0.1
0.01
100***
100***
100***
76***
NI
0.17 0.01
3. Conclusion
Novel and non-natural N7 purine nucleosides, as well as some of
their bicyclic sugar counterparts demonstrated a high potential for
the selective inhibition of butyrylcholinesterase, with IC50 values of
the same order of magnitude as that of the positive control rivastig-
mine. Interestingly, for the nucleobase-free derivatives the presence
of benzyl protecting groups and the azido group seem to be signifi-
cantforBChEinhibition. Regardingnucleosidessynthesis, anefficient
regioselective N7-glycosylation procedure of the 2-acetamido-6-
chloropurinewasdevelopedstartingfrom1-acetoxyglycosyldonors,
in acetonitrile at 65 °C and using TMSOTf as promoter. Noteworthy,
the increase of the number of equivalents of the Lewis acid and reac-
tion time (3.5 h) allowed the direct coupling of a methyl glucopyran-
oside with a 2-acetamido purine base, in reasonable yields. To the
best of our knowledge, this is the first direct synthesis of a nucleoside
startingfrom anunactivated methyl glycopyranoside. Thelowvalues
for acute cytotoxicity of the probe molecules tested encourage fur-
ther investigation of this family of compounds for the control of neu-
rodegenerative disorders such as Alzheimer’s disease.
a
IC50 is an estimate of the compound concentration which inhibits 50% of the
enzyme activity; values are expressed as mean standard error of the mean (SEM).
b
Rivastigmine is a standard drug to treat AD patients and was used as positive
control. ***P < 0.001.
Na/benzophenone (THF) or P2O5 [CH2Cl2, (CH2Cl)2]. Other solvents
were dried over a bed of activated molecular sieves (MS) 4 Å (DMF)
or KOH (pyridine). Solvents for column chromatography (cyclohex-
ane, EtOAc, CH2Cl2) were distilled before use. Reactions were mon-
itored by thin-layer chromatography (TLC) on a precoated plate of
Silica Gel 60 F254 (layer thickness 0.2 mm; E. Merck, Darmstadt,
Germany) and detection by UV light (254 nm) and by charring with
H2SO4 10% in EtOH or with 0.2% w/v cerium sulfate and 5% ammo-
nium molybdate in 2 M H2SO4. Flash column chromatography was
performed on silica gel 60 (230–400 mesh, E. Merck). Melting
points (mp) were determined with a Büchi B-510 capillary appara-
tus and are uncorrected. Optical rotations ([a]D) were measured at
20 2 °C with a Perkin Elmer Model 241 digital polarimeter, using
a 10 cm, 1 mL cell. High resolution mass spectra (HRMS) using
chemical ionization (CI, ammonia) or fast atom bombardment
(FAB) were obtained with a JMS-700 spectrometer at École Nor-
male Supérieure de Paris (ENS-Paris). Elemental analyses were per-
formed by the service d’analyse of UPMC-Paris VI. NMR
experiments were recorded on Brüker spectrometers: 1H NMR
spectra were recorded at 400 MHz with a DRX 400 at ENS-Paris,
4. Experimental
4.1. Chemistry
4.1.1. General methods
Solvents and reagents were purchased from Fluka, Aldrich or
Acros Organics. Solvents were freshly distilled under argon from

F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
5111
Table 3
4.1.2. Methyl 3-C,2-O-[(1S)-1-hydroxyethylene]-a-D-
glucopyranoside (2)
Preliminary screening of the acute toxicity of compounds 7a, 8a, 8b, 14a, 15a17 and
15b18 in immortalized human fibroblasts (A549 cells)
The bicyclic derivative 1 (0.73 g, 2.25 mmol) was dissolved in
80% aqueous solution of acetic acid (25 mL) and warmed to 50 °C
over a period of 2 h. The solvent was removed under reduced pres-
sure and the residue obtained co-evaporated with toluene
(3 ꢁ 50 mL). Purification by flash column chromatography
(CH2Cl2/MeOH 8:2) afforded the miharamycins bicyclic unit 2
(0.53 g, quant. yield) as a white solid. Rf 0.23 (EtOAc/MeOH, 9:1);
Compound no.
Concentration (
mL)
l
g/ Cell viability SEM (% of
control)
COOBn
(R)
HO
O
O
BnO
2000
200
20
35.95 3.84
63.58 10.37
67.24 12.68
81.56 12.49
91.88 13.98
BnO
BnO
+125.3 (c 1.0, MeOH); 1H
OMe
mp 142–144 °C (CH2Cl2/MeOH); [
a]
D
7a
NMR (D2O, 400 MHz): d 4.95 (d, 1H, J1,2 = 5.4 Hz, H-1), 4.36 (dd,
1H, J8a,7 = 5.0 Hz, J8a,8b = 10.1 Hz, H-8a), 4.25 (d, 1H, J7,8a = 5.0 Hz,
H-7), 4.04 (d, 1H, J2,1 = 5.4 Hz, H-2), 3.93 (d, 1H, J 4,5 = 10.0 Hz, H-
4), 3.93–3.83 (m, 3H, H-5, H-6a, H-8b), 3.72 (dd, 1H, J6b,5 = 5.5 Hz,
J6b,6a = 12.3 Hz, H-6b), 3.46 (s, 3H, OCH3); 13C NMR (D2O,
100 MHz): d 98.13 (C-1), 82.30 (C-3), 81.74 (C-2), 77.29 (C-8),
76.78 (C-7), 71.68 (C-5), 70.75 (C-4), 61.15 (C-6), 55.62 (OCH3);
HRMS (CI) m/z: [M+NH4]+ calcd for C9H20O7N, 254.1234, found:
254.1241; Anal. Calcd for C9H16O7: C, 45.76; H, 6.83. Found: C,
45.80; H, 6.90.
2
0.2
COOBn
(S)
N3
O
BnO
2000
200
20
24.97 2.49
47.94 3.84
73.90 16.91
71.23 10.38
90.22 9.42
BnO
BnO
O
OMe
8a
2
0.2
COOBn
(R)
N3
O
BnO
BnO
BnO
4.1.3. Methyl 3,4,6-tri-O-acetyl-3-C,2-O-[(1S)-1-
(acetoxy)ethylene]-a-D-glucopyranoside (3)
2000
200
20
15.98 4.61
64.25 17.87
63.58 21.72
76.23 10.18
90.54 15.75
O
OMe
To the bicyclic compound 2 (0.27 g, 1.14 mmol) in dry pyridine
(5 mL) were added acetic anhydride (2.5 mL) and a catalytic
amount of DMAP. The reaction mixture was stirred overnight at
room temperature, then co-evaporated with toluene and concen-
trated. The crude residue was dissolved in CH2Cl2 (80 mL) and
washed with a 1 M aqueous solution of HCl (20 mL). The organic
layer was separated, dried (MgSO4) and concentrated under re-
duced pressure. Purification by flash column chromatography
(cyclohexane/EtOAc, 1:1) afforded the peracetylated compound 3
(0.43 g, 92%) as a colorless oil. Rf 0.32 (cyclohexane/EtOAc, 1:1);
8b
2
0.2
COOBn
N3
(
)
N
S
NHAc
NHAc
NHAc
N
N
O
BnO
N
2000
200
20
7.32 1.92
51.26 4.61
46.94 16.72
82.22 2.88
88.54 3.46
BnO
BnO
Cl
O
14a
2
0.2
[a]
+95.5 (c 0.7, CHCl3); 1H NMR (CDCl3, 400 MHz): d 5.91 (d,
D
COOMe
N3
(
)
N
S
N
N
1H, J4,5 = 10.3 Hz, H-4), 5.51 (dd, 1H, J7,8b = 2.2 Hz, J7,8a = 5.7 Hz,
H-7), 4.92 (d, 1H, J1,2 = 5.2 Hz, H-1), 4.77 (d, 1H, J2,1 = 5.2 Hz, H-2),
4.22 (ddd, 1H, J5,6b = 2.4 Hz, J5,6a = 5.4 Hz, J5,4 = 10.3 Hz, H-5),
4.17–4.13 (m, 2H, H-6, H-8a), 4.04 (dd, 1H, J6b,5 = 2.4 Hz,
J6b,6a = 12.6 Hz, H-6b), 3.91 (dd, 1H, J8b,7 = 2.2 Hz, J8b,8a = 10.0 Hz,
H-8b), 3.36 (s, 3H, OCH3), 2.03, 1.99, 1.94, 1.93 (4 ꢁ s, 12H,
4 ꢁ OAc); 13C NMR (CDCl3, 100 MHz): d 170.31, 169.92, 169.55,
169.12 (4 ꢁ C@O), 98.20 (C-1), 89.49 (C-3), 79.03 (C-2), 76.58 (C-
7), 73.18 (C-8), 66.22 (C-5), 65.93 (C-4), 62.41 (C-6), 55.12
(OCH3), 21.74, 20.73, 20.35, 20.16 (4 ꢁ OAc); HRMS (FAB) m/z:
[M+Na]+ calcd for C17H28O11Na, 422.1662, found: 422.1671.
O
BnO
N
2000
200
20
15.05 3.56
19.54 0.81
40.84 0.18
77.70 5.88
102.90 0.84
BnO
BnO
Cl
15a17
O
2
0.2
COOMe
N3
N
N
Cl
(R)
N
N
O
BnO
2000
200
20
21.27 15.16
52.27 3.62
69.27 4.29
99.90 0.50
102.91 2.80
BnO
BnO
O
15b18
2
0.2
4.1.4. Methyl 3,4,6-tri-O-benzoyl-3-C,2-O-[(1S)-1-
(benzoyloxy)ethylene]-a-D-glucopyranoside (4)
To the bicyclic compound 2 (0.16 g, 0.67 mmol) in anhydrous
pyridine (2.5 mL) were added benzoyl chloride (0.78 mL,
6.71 mmol) and a catalytic amount of DMAP. The reaction mixture
was stirred over 8 h at room temperature and then warmed to
80 °C. After stirring for 48 h, TLC revealed no trace of starting mate-
rial; then the reaction mixture was co-evaporated with toluene
and concentrated. The crude residue was dissolved in CH2Cl2
(80 mL) and washed with a 1 M aqueous solution of HCl (20 mL).
The organic layer was separated, dried (MgSO4) and concentrated
under reduced pressure. Purification by flash column chromatogra-
phy (cyclohexane/EtOAc, 4:1) afforded the perbenzoylated com-
pound 4 (0.32 g, 72%) as a colorless oil. Rf 0.58 (cyclohexane/
DMSO
2000
200
20
60.25 11.01
89.54 0.58
86.22 5.19
104.19 13.64
90.54 6.15
44.51 0.65
89.02 5.76
100.00 7.39
2
0.2
H2O2% (positive control)
Rivastigmine, 100
Blank sample
lg/mL
or with an Avance 400 (fitted with a QNP probe) at FCUL for soln in
CDCl3, or D2O at room temperature. Assignments were confirmed
by COSY experiments. 13C NMR spectra were recorded at
100 MHz with a DRX 400 spectrometer at ENS-Paris, or with an
Avance 400 (fitted with a QNP probe) at FCUL. Assignments were
confirmed by J-mod technique, HMQC and HMBC. Chemical shifts
(d) are given in ppm relative to tetramethylsilane (TMS) or to the
residual solvent signal. Coupling constants (J) are reported in hertz
(Hz).
EtOAc, 1:1); [
8.23–7.26 (m, 20H, H arom.), 6.83 (d, 1H, J4,5 = 10.3 Hz, H-1),
a]
+105 (c 0.8, CHCl3); 1H NMR (CDCl3, 400 MHz):
D
d
6.09 (dd, 1H, J7,8b = 1.8 Hz, J7,8a = 5.3 Hz, H-7), 5.31 (d, 1H,
J2,1 = 5.4 Hz, H-2), 5.27 (d, 1H, J1,2 = 5.4 Hz, H-1), 4.85 (ddd, 1H,
J5,6a = 3.2 Hz, J5,6b = 5.3 Hz, J5,4 = 10.3 Hz, H-5), 4.61 (dd, 1H,
J6a,5 = 3.2 Hz, J6a,6b = 12.0 Hz, H-6a), 4.56 (dd, 1H, J8a,7 = 5.3 Hz,

5112
F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
J8a,8b = 10.3 Hz, H-8a), 4.43 (dd, 1H, J6b,5 = 5.3 Hz, J6b,6a = 12.0 Hz, H-
6b), 4.29 (dd, 1H, J8b,7 = 1.8 Hz, J8b,8a = 10.3 Hz, H-8b), 3.64 (s, 3H,
OCH3); 13C NMR (CDCl3, 100 MHz): d 166.51, 165.97, 165.90,
165.76 (4x C@O), 134.11–133.46 (5CH arom.), 130.60, 130.57,
130.03, 129.14 (4C arom. quat.), 130.31–128.70 (15CH arom.),
99.42 (C-1), 91.20 (C-3), 79.92 (C-2), 78.54 (C-7), 74.92 (C-8),
68.22 (C-4), 67.20 (C-5), 64.34 (C-6), 56.31 (OCH3); HRMS (FAB)
m/z: [M+Na]+ calcd for C37H32O11Na, 675.1842, found: 675.1849.
4.26 (dd, 1H, J8,9a = 5.4 Hz, J8,9b = 1.5 Hz, H-8), 4.19 (dd, 1H,
J9b,8 = 1.5 Hz, J9b,9a = 8.8 Hz, H-9b), 3.50 (s, 3H, OCH3), 3.29 (d, 1H,
JOH,6 = 3.7 Hz, OH); 13C NMR (CDCl3, 100 MHz): d 172.29 (C@O),
139.04, 138.19, 137.93, 135.05 (4C arom. quat.), 128.59 -126.80
(20CH arom.), 99.34 (C-1), 90.06 (C-3), 85.63 (C-8), 77.28 (C-2),
75.34 (C-9), 74.37 (CH2Ph), 72.80 (C-4), 72.72 (CH2Ph), 70.52 (C-
6), 69.97 (C-5), 67.32 (CH2Ph), 64.49 (CH2Ph), 55.31 (OCH3); HRMS
(CI) m/z: [M+NH4]+ calcd for C38H44O9N, 658.3016, found:
658.3010; Anal. Calcd for C38H44NO9: C, 71.23; H, 6.29. Found: C,
70.98; H, 6.18.
4.1.5. Benzyl {methyl 3,4-di-O-benzyl-3-C,2-O-[(1S)-1-
(benzyloxy)ethylene]-6-hydroxy-
heptopyranosid}uronate (7a) and benzyl {methyl-3,4-di-O-
benzyl-3-C,2-O-[(1S)-1-(benzyloxy)ethylene]-6-hydroxy-
glycero- -gluco-heptopyranosid}uronate (7b)
L-glycero-a-D-gluco-
4.1.6. Benzyl {methyl 6-azido-3,4-di-O-benzyl-3-C,2-O-[(1S)-1-
D-
(benzyloxy)ethylene]-6-deoxy-D-glycero-a-D-gluco-
a-D
heptopyranosid}uronate (8a)
A mixture of both epimeric allylic alcohols 6a and 6b (ratio 1:1,
0.36 g, 0.67 mmol) was dissolved in CH2Cl2 (80 mL) and cooled to
ꢀ78 °C. Ozone was bubbled through the reaction mixture until
persistence of a blue color (typically 5 min). Dimethylsulfide
(0.1 mL) was added and the reaction mixture was allowed to reach
the room temperature. The solvent was removed under reduced
pressure and the mixture of epimeric aldehydes was used in the
next step without further purification. NaH2PO4.H2O (1.20 g,
8.71 mmol) and NaClO2 (1.09 g, 12.06 mmol) were added to a solu-
tion of the crude aldehydes in 2-methylbut-2-ene (1.5 mL), tBuOH
(3 mL) and water (3 mL). After stirring overnight at room temper-
ature the reaction mixture was diluted with EtOAc (50 mL) and
water (20 mL). The aqueous layer was extracted with EtOAc
(3 ꢁ 50 mL). The organic layers were combined, dried (MgSO4), fil-
tered and concentrated. The carboxylic acids obtained were di-
rectly engaged in the next step. To a solution of both epimeric
carboxylic acids in DMF (8 mL) was added sequentially Bu4NI
(0.99 g, 2.68 mmol) and KHCO3 (0.37 g, 3.68 mmol) followed by
slowly addition of benzyl bromide (0.32 mL, 2.68 mmol). After stir-
ring overnight the solvent was removed under reduced pressure
and the crude residue diluted with Et2O (80 mL) and water
(30 mL). The aqueous layer was extracted with ether (3 ꢁ 80 mL),
the organic layers were combined, dried (MgSO4), filtered and con-
centrated under reduced pressure. Purification by flash column
chromatography (cyclohexane/EtOAc, 5:1 then cyclohexane/EtOAc,
3:1) afforded the benzyl ester 6R 7a (154 mg, 36% over three steps)
To a solution of benzyl ester 7a (175 mg, 0.27 mmol) in dry
CH2Cl2 (5.5 mL), anhydrous pyridine (0.35 mL, 4.37 mmol) was
added followed by slow addition of Tf2O (0.37 mL, 2.19 mmol) at
ꢀ78 °C under argon. After stirring 2 h at room temperature, water
(10 mL) and CH2Cl2 (20 mL) were added. The organic layer was
separated, dried (MgSO4), filtered and concentrated under reduced
pressure. The crude triflate compound was directly engaged in the
next step. Sodium azide (106 mg, 1.64 mmol) was added to a solu-
tion of the crude triflate product in anhydrous DMF (6 mL) at room
temperature. After stirring overnight the reaction mixture was
concentrated under reduced pressure. The residue was then di-
luted with ether (80 mL) and water (30 mL). The aqueous layer
was extracted with ether (3 ꢁ 80 mL), the organic layers were
combined, dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (cyclohex-
ane/EtOAc, 5:1) afforded the benzyl azido ester 6S 8a (131 mg, 72%
over two steps) as a colorless oil. Rf 0.66 (cyclohexane/EtOAc 1:1);
[a]
+85.0 (c 0.6, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.42–7.28
D
(m, 20H, H arom.), 5.08 (d, 1H, J = 12.1 Hz, CHPh), 5.00 (d, 1H,
J1,2 = 5.8 Hz, H-1), 4.98 (dd, 1H, J5,6 = 1.7 Hz, J5,4 = 10.1 Hz, H-5),
4.85 (d, 1H, J = 12.1 Hz, CHPh), 4.69 (d, 1H, J = 10.5 Hz, CHPh),
4.61 (d, 1H, J = 12.4 Hz, CHPh), 4.53 (s, 2H, CH2Ph), 4.52 (d, 1H,
J = 12.4 Hz, CHPh), 4.40 (d, 1H, J6,5 = 1.7 Hz, H-6), 4.34 (d, 1H,
J2,1 = 5.8 Hz, H-2), 4.35 (d, 1H, J4,5 = 10.1 Hz, H-4), 4.34–2.28 (m,
2H, H-8, H-9a), 4.23 (dd, 1H, J9b,8 = 1.4 Hz, J9b,9a = 8.8 Hz, H-9b),
3.56 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz): d 167.49 (C@O),
138.79, 138.12, 137.74, 134.86 (4C arom. quat.), 128.64–126.86
(20CH arom.), 99.47 (C-1), 89.60 (C-3), 85.40 (C-8), 77.11 (C-2),
75.27 (C-9), 74.39 (CH2Ph), 74.08 (C-4), 72.74 (CH2Ph), 69.71 (C-
5), 66.98 (CH2Ph), 64.49 (CH2Ph), 62.49 (C-6), 55.69 (OCH3); HRMS
(CI) m/z: [M+NH4]+ calcd for C38H43O8N4, 683.3081, found:
683.3073.
as a colorless oil. Rf 0.61 (cyclohexane/EtOAc 2:1); [a] +76 (c 1.0,
D
CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.38–7.22 (m, 20H, H arom.),
5.36 (d, 1H, J = 12.2 Hz, CHPh), 5.13 (d, 1H, J = 12.2 Hz, CHPh), 4.99
(d, 1H, J = 11.2 Hz, CHPh), 4.96 (d, 1H, J = 11.8 Hz, CHPh), 4.89 (d,
1H, J1,2 = 5.8 Hz, H-1), 4.82 (d, 1H, J = 11.8 Hz, CHPh), 4.78 (dd,
1H, J5,6 = 1.7 Hz, J5,4 = 10.0 Hz, H-5), 4.69 (d, 1H, J = 11.8 Hz, CHPh),
4.67 (d, 1H, J = 11.2 Hz, CHPh), 4.60 (d, 1H, J6,5 = 1.7 Hz, H-6), 4.57
(d, 1H, J = 11.8 Hz, CHPh), 4.39 (d, 1H, J4,5 = 10.0 Hz, H-4), 4.35–
4.31 (m, 3H, H-2, H-8, H-9a), 4.21 (dd, 1H, J9b,8 = 4.1 Hz,
J9b,9a = 7.2 Hz, H-9b), 3.16 (s, 3H, OCH3); 13C NMR (CDCl3,
100 MHz): d 173.10 (C@O), 138.86, 138.56, 138.16, 134.82 (4C
arom. quat.), 129.58–126.98 (20CH arom.), 99.53 (C-1), 88.82 (C-
3), 85.31 (C-2 or C-8), 77.82 (C-2 or C-8), 75.19 (C-9), 74.76
(CH2Ph), 74.38 (C-4), 72.95 (CH2Ph), 69.60 (C-6), 69.32 (C-5),
67.45 (CH2Ph), 64.74 (CH2Ph), 55.01 (OCH3); HRMS (CI) m/z:
[M+NH4]+ calcd for C38H44O9N, 658.3016, found: 658.3022; Anal.
Calcd for C38H44NO9: C, 71.23; H, 6.29. Found: C, 69.27; H, 6.41.
Further elution afforded the benzyl ester 6S 7b (140 mg, 32%, over
4.1.7. Benzyl {methyl 6-azido-3,4-di-O-benzyl-3-C,2-O-[(1S)-1-
(benzyloxy)ethylene]-6-deoxy-
heptopyranosid}uronate (8b)
L-glycero-a-D-gluco-
To a solution of benzyl ester 7b (137 mg, 0.21 mmol) in dry
CH2Cl2 (5 mL), anhydrous pyridine (0.28 mL, 3.42 mmol) was
added followed by slow addition of Tf2O (0.29 mL, 1.71 mmol) at
ꢀ78 °C under argon. After stirring 2 h at room temperature, water
(10 mL) and CH2Cl2 (20 mL) were added. The organic layer was
separated, dried (MgSO4), filtered and concentrated under reduced
pressure. The crude triflate compound was directly engaged in the
next step. Sodium azide (83 mg, 1.28 mmol) was added to a solu-
tion of the crude triflate product in anhydrous DMF (5 mL) at room
temperature. After stirring overnight the reaction mixture was
concentrated under reduced pressure. The residue was then di-
luted with ether (80 mL) and water (30 mL). The aqueous layer
was extracted with ether (3 ꢁ 80 mL), the organic layers were
combined, dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (cyclohex-
ane/EtOAc, 5:1) afforded the benzyl azido ester 6R 8b (122 mg, 86%
three steps) as a colorless oil. Rf 0.51 (cyclohexane/EtOAc 2:1); [a]
D
+50.5 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.38–7.15 (m,
20H, H arom.), 5.02 (d, 1H, J = 12.0 Hz, CHPh), 4.99 (d, 1H,
J1,2 = 5.8 Hz, H-1), 4.96 (d, 1H, J = 10.1 Hz, CHPh), 4.83 (dd, 1H,
J5,6 = 2.1 Hz, J5,4 = 10.2 Hz, H-5), 4.58 (d, 1H, J = 10.1 Hz, CHPh),
4.56 (s, 2H, CH2Ph), 4.55 (d, 1H, J = 12.5 Hz, CHPh), 4.40 (m, 2H,
H-6, CHPh), 4.40 (d, 1H, J4,5 = 10.2 Hz, H-4), 4.35 (d, 1H,
J2,1 = 5.8 Hz, H-2), 4.31 (dd, 1H, J9a,8 = 5.4 Hz, J9a,9b = 8.8 Hz, H-9a),

F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
5113
over two steps) as a colorless oil. Rf 0.68 (cyclohexane/EtOAc 1:1);
+19.1 (c 0.2, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.42–7.26
4.1.9. 2-(Acetylamino)-6-chloro-9-{6-O-acetyl-3,4-di-O-benzyl-
3-C,2-O-[(1S)-1-benzyloxyethylene]– -glucopyranosyl}purine
(11) and 2-(acetylamino)-6-chloro-7-{6-O-acetyl-3,4-di-O-
benzyl-3-C,2-O-[(1S)-1-benzyloxyethylene]–D-
glucopyranosyl}purine (12)
[
a]
D
D
(m, 20H, H arom.), 5.34 (d, 1H, J = 12.2 Hz, CHPh), 5.19 (d, 1H,
J = 12.2 Hz, CHPh), 5.05 (d, 1H, J = 11.3 Hz, CHPh), 5.02 (dd, 1H,
J5,6 = 2.2 Hz, J5,4 = 10.4 Hz, H-5), 4.92 (d, 1H, J1,2 = 5.9 Hz, H-1),
4.74 (d, 1H, J = 11.3 Hz, CHPh), 4.70 (d, 1H, J = 12.0 Hz, CHPh),
4.69 (s, 2H, CH2Ph), 4.58 (d, 1H, J = 12.0 Hz, CHPh), 4.38 (d, 1H,
J2,1 = 5.9 Hz, H-2), 4.37–4.33 (m, 2H, H-8, H-9a), 4.31 (d, 1H,
J6,5 = 2.2 Hz, H-6), 4.28 (d, 1H, J4,5 = 10.4 Hz, H-4), 4.24 (dd, 1H,
J9b,8 = 4.6 Hz, J9b,9a = 9.8 Hz, H-9b), 3.19 (s, 3H, OCH3); 13C NMR
BSA (70.7
lL, 286
lmol) was added to a suspension of the 2-
acetamido-6-chloropurine (30.3 mg, 143
lmol) in anhydrous 1,2-
dichloroethane (1 mL) under argon. The mixture was heated to
80 °C for 45 min. To this solution was added a solution of the gly-
cosyl donor 9 (55 mg, 95.4
was added, followed by slow addition of TMSOTf (138.1
763 mol) under argon. The reaction mixture was stirred at 85 °C
l
mol) in dry 1,2-dichloroethane (1 mL)
(CDCl3, 100 MHz):
d
168.80 (C@O), 138.68, 138.16, 137.95,
lL,
134.81 (4C arom. quat.), 128.53–127.06 (20CH arom.), 99.47 (C-
1), 89.56 (C-3), 85.30 (C-8), 77.56 (C-2), 75.24 (C-9), 75.21 (C-4),
74.90 (CH2Ph), 72.96 (CH2Ph), 69.49 (C-5), 67.53 (CH2Ph), 64.79
(CH2Ph), 61.37 (C-6), 55.23 (OCH3); HRMS (CI) m/z: [M+NH4]+ calcd
for C38H43O8N4, 683.3081, found: 683.3076.
l
for 4 h, cooled to room temperature, diluted with CH2Cl2 (10 mL).
The organic layer was neutralized with a NaHCO3 saturated aque-
ous solution (2 ꢁ 5 mL). The aqueous layer was extracted with
CH2Cl2 (3 ꢁ 15 mL), and the organic layers were combined, dried
(MgSO4), filtered and concentrated under reduced pressure. Purifi-
cation by flash column chromatography (cyclohexane/EtOAc, 2:1
then cyclohexane/EtOAc, 1:1) afforded the N9 regioisomer 11
(17 mg, 25%) as a colorless oil. Rf 0.63 (cyclohexane/EtOAc, 1:1);
4.1.8. 1,6-Di-O-acetyl-3,4-di-O-benzyl-3-C,2-O-[(1S)-1-
(benzyloxy)ethylene]-b-D-glucopyranose (9)
Sodium hydride (275 mg, 6.86 mmol, 60% w/w) was added to a
solution of the bicyclic compound 2 (270 mg, 1.14 mmol) in anhy-
drous DMF (8 mL) at 0 °C. After 30 min, BnBr (1.1 mL, 9.15 mmol)
wasaddedat0 °C. Afterstirringfor2.5 hat roomtemperature, MeOH
(10 mL) was added and the mixture was concentrated under re-
duced pressure. The residue was then diluted with ether (80 mL)
and water (30 mL). The aqueous layer was extracted with ether
(3 ꢁ 80 mL), the organic layers were combined, dried (MgSO4), fil-
tered and concentratedunder reduced pressure. Purification by flash
column chromatography (cyclohexane then cyclohexane/EtOAc,
4:1) afforded the fully protected benzylated compound (579 mg,
[a
]
D
+18.2 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.14 (s,
1H, H-8), 8.05 (br s, 1H, NH), 7.42–7.29 (m, 15H, H arom.), 6.10
(d, 1H, J1 ,2 = 8.9 Hz, H-10), 5.00 (m, 2H, H-20, CHPh), 4.82–4.64
(m, 5H, CHPh, 2 ꢁ CH2Ph), 4.47–4.38 (m, 3H, H-50, H-70, H-60a),
4.29–4.21 (m, 3H, H-40, H-60b, H-80a), 4.12 (app d, 1H,
0
0
J8 b,8 a = 9.5 Hz, H-80b), 2.49 (s, 3H, NHAc), 2.05 (s, 3H, OAc); 13C
NMR (CDCl3, 100 MHz): d 170.51, 171.06 (2 ꢁ C@O), 153.01 (C-4),
152.49 (C-2 or C-6), 152.00 (C-2 or C-6), 143.35 (C-8), 137.94,
137.70, 137.39 (3C arom. quat.), 129.15–127.63 (15CH arom.),
128.70 (C-5), 90.02 (C-30), 85.19 (C-70), 83.31 (C-10), 77.58 (C-20),
76.27 (C-50), 75.32 (CH2Ph), 74.58 (C-80), 74.46 (C-40), 73.97
(CH2Ph), 65.73 (CH2Ph), 63.85 (C-60), 25.62 (NHAc), 21.29 (OAc);
HRMS (CI) m/z: [M+H]+ calcd for C38H39O8N535Cl, 728.2487, and
C38H39O8N537Cl, 730.2458, found: 728.2515 and 730.2465, respec-
tively. Further elution (cyclohexane/EtOAc, 1:2 then cyclohexane/
EtOAc, 1:4) afforded the N7 regioisomer 12 (26 mg, 37%) as a color-
0
0
85%) as a colorless oil. Rf 0.56 (cyclohexane/EtOAc, 2:1); [
+115.8 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.54–7.43 (m,
20H, arom.), 5.18 (d, 1H, J1,2 = 5.8 Hz, H-1), 5.07 (d, 1H,
a]
D
H
J = 11.2 Hz, CHPh), 4.86–4.77 (m, 5H, 3 ꢁ CHPh, CH2Ph), 4.72–4.67
(m, 3H, 2 ꢁ CHPh, H-5), 4.56 (d, 1H, J2,1 = 5.8 Hz, H-2), 4.53–4.42
(m, 4H, H-4, H-7, H-8a, H-8b), 3.99 (dd, 1H, J 6a,5 = 3.6 Hz, J
6a,6b = 10.8 Hz, H-6a), 3.90 (dd, 1H, J6b,5 = 1.9 Hz, J6b,6a = 10.8 Hz, H-
6b), 3.85 (s, 3H, OCH3); 13C NMR (CDCl3, 100 MHz): d 138.73,
138.39, 138.12, 137.66 (4C arom. quat.), 128.61–126.63 (20CH
arom.), 98.86 (C-1), 89.38 (C-3), 84.96 (C-2), 77.90 (C-7), 74.76 (C-
8), 74.57 (C-4), 74.08 (CH2Ph), 72.98 (CH2Ph), 72.36 (CH2Ph), 68.55
(C-6), 67.48 (C-5), 64.37 (CH2Ph), 54.60 (OCH3); HRMS (FAB) m/z:
[M+Na]+ calcd for C37H40O7Na, 619.2666, found: 619.2668; Anal.
Calcd for C37H40O7: C, 74.47; H, 6.76. Found: C, 74.45; H, 6.69. To a
solution of the perbenzylated derivative (450 mg, 0.76 mmol) in
acetic anhydride (15 mL) was added dropwise sulfuric acid 5% in
AcOH (0.4 mL) at ꢀ20 °C. The reaction mixture was stirred at this
temperature over 25 min and then neutralized by slowly addition
of a NaHCO3 saturated aqueous solution. The organic layer was sep-
arated, and the aqueous layer was extracted with CH2Cl2
(3 ꢁ 150 mL). The organic layers were combined, dried (MgSO4), fil-
tered and concentrated. Purification by flash column chromatogra-
phy (cyclohexane/CH2Cl2/EtOAc, 8:1:1) afforded the suitable
glycosyl donor 9 (209 mg, 48%) as a colorless oil. Rf 0.54 (cyclohex-
less oil. Rf 0.14 (cyclohexane/EtOAc, 1:1); [a]D +28.8 (c 1.0, CHCl3);
1H NMR (CDCl3, 400 MHz): d 8.47 (s, 1H, H-8), 8.37 (br s, 1H, NH),
7.41–7.29 (m, 15H, H arom.), 6.45 (d, 1H, J1 ,2 = 9.1 Hz, H-10), 5.00
(d, 1H, J = 10.9 Hz, CHPh), 4.80–4.60 (m, 6H, H-20, CHPh,
2 ꢁ CH2Ph), 4.45–4.41 (m, 3H, H-50, H-60a, H-70), 4.34 (dd, 1H,
0
0
J8 a,7 = 3.3 Hz, J8 a,8 b = 9.4 Hz, H-80a), 4.24 (dd, 1H, J6 b,5 = 5.6 Hz,
0
0
0
0
0
0
J6 b,6 a = 12.6 Hz, H-60b), 4.21–4.10 (m, 2H, H-40, H-80b), 2.60 (s,
3H, NHAc), 2.06 (s, 3H, OAc); 13C NMR (CDCl3, 100 MHz): d
171.50, 170.63 (2 ꢁ C@O), 163.18 (C-4), 152.53 (C-2 or C-6),
146.36 (C-8), 143.95 (C-2 or C-6), 137.50, 137.11, 136.80 (3C arom.
quat.), 128.71–127.24 (15CH arom.), 118.97 (C-5), 89.73 (C-30),
84.56 (C-70), 82.95 (C-10), 77.55 (C-20), 75.74 (C-50), 74.80 (CH2Ph),
74.37 (C-80), 73.86 (C-40), 73.60 (CH2Ph), 65.37 (CH2Ph), 63.23 (C-
60), 25.26 (NHAc), 20.89 (OAc); HRMS (FAB) m/z: [M+Na]+ calcd
for C38H38O8N535ClNa, 750.2307, and C38H38O8N537ClNa,
752.2277, found: 750.2310 and 752.2309, respectively.
0
0
4.1.10. Benzyl1-O-acetyl-6-azido-3,4-di-O-benzyl-3-C,2-O-[(1S)-
ane/CH2Cl2/EtOAc, 2:1:1); [
a
]
D +33.9 (c 1.0, CHCl3); 1H NMR (CDCl3,
1-(benzyloxy)ethylene]-6-deoxy-D-glycero–D-gluco-hepto-
400 MHz): d7.40–7.28 (m, 15H, H arom.), 6.10 (d, 1H, J1,2 = 7.8 Hz, H-
1), 4.91 (d, 1H, J = 11.0 Hz, CHPh), 4.71–4.60 (m, 5H, CHPh,
2 ꢁ CH2Ph), 4.38–4.25 (m, 5H, H-2, H-5, H-7, H-6a, H-6b), 4.20 (dd,
1H, J8a,7 = 3.5 Hz, J8a,8b = 9.3 Hz, H-8a), 4.10–4.04 (m, 2H, H-4, H-
8b), 2.18, 2.09 (2 ꢁ s, 6H, OAc); 13C NMR (CDCl3, 100 MHz): d
171.22, 169.78 (2 ꢁ C@O), 138.27, 137.97, 137.75 (C arom. quat.),
128.99–127.72 (15CH arom.), 93.90 (C-1), 90.35 (C-3), 84.84 (C-2
or C-7), 78.36 (C-2 or C-7), 75.03 (CH2Ph), 74.33 (C-4), 74.01 (C-8),
73.89 (C-5), 73.25 (CH2Ph), 65.39 (CH2Ph), 63.80 (C-6), 21.57
(OAc), 21.35 (OAc); HRMS (FAB) m/z: [M+Na]+ calcd for C33H36O9Na,
599.2257, found: 599.2252.
pyranuronate (13a)
A solution of concd sulfuric acid (5% in AcOH, 70
dropwise at ꢀ20 °C to a solution of benzyl azido ester 8a (170 mg,
255 mol) in acetic anhydride (6 mL). The reaction mixture was al-
lL) was added
l
lowed to warm to 0 °C over a period of 15 min, and then neutral-
ized by slow addition of a NaHCO3 saturated aqueous solution.
The organic layer was separated, and the aqueous layer was ex-
tracted with CH2Cl2 (3 ꢁ 50 mL). The organic layers were com-
bined, dried (MgSO4), filtered and concentrated. Purification by
flash column chromatography (cyclohexane/CH2Cl2/EtOAc,
10:1:1) afforded the glycosyl donor 13a (64 mg, 36%) as a colorless

5114
F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
oil. Rf 0.46 (cyclohexane/CH2Cl2/EtOAc, 6:1:1); [
a
]
+23.8 (c 0.2,
was added the glycosyl donor 6S 13a (30 mg, 43.2
in anhydrous CH3CN (1 mL) followed by slow addition of TMSOTf
(156 L, 864 mol) under argon. The solution was stirred at 65 °C
for 90 min. The mixture was cooled to room temperature, CH2Cl2
(20 mL) was added and the organic layer was washed with NaHCO3
saturated aqueous solution (2 ꢁ 10 mL). The aqueous layer was ex-
tracted with CH2Cl2 (3 ꢁ 30 mL), the organic layers were com-
bined, dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (cyclohex-
ane/EtOAc, 1:1) afforded the N7 nucleoside 6S 14a (22 mg, 60%) as
lmol) dissolved
D
CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.50–7.00 (m, 20H, H arom.),
6.13 (d, 1H, J1,2 = 7.8 Hz, H-1), 5.01 (d, 1H, J = 12.0 Hz, CHPh), 4.99
(d, 1H, J = 9.8 Hz, CHPh), 4.79 (dd, 1H, J5,6 = 2.4 Hz, J5,4 = 9.8 Hz,
H-5), 4.74 (d, 1H, J = 12.0 Hz, CHPh), 4.64–4.58 (m, 3H, CH2Ph,
CHPh), 4.51 (d, 1H, J6,5 = 2.4 Hz, H-6), 4.45–4.42 (m, 3H, H-4,
CH2Ph), 4.26 (d, 1H, J2,1 = 7.8 Hz, H-2), 4.21–4.16 (m, 2H, H-8, H-
9a), 3.95 (d, 1H, J9b,9a = 9.0 Hz, H-9b), 2.21 (s, 3H, OAc); 13C NMR
(CDCl3, 100 MHz): d 169.37, 169.09 (2 ꢁ C@O), 137.71, 137.33,
137.19, 134.58 (4C arom. quat.), 128.70–127.45 (20CH arom.),
93.29 (C-1), 89.98 (C-3), 84.25 (C-8), 77.31 (C-2), 74.98 (C-5),
74.56 (CH2Ph), 74.03 (C-9), 72.85 (C-4), 72.74 (CH2Ph), 67.64
(CH2Ph), 64.84 (CH2Ph), 62.85 (C-6), 21.05 (OAc); HRMS (CI) m/z:
[M+NH4]+ calcd for C39H43O9N4, 711.3030, found: 711.3025.
l
l
a colorless oil. Rf 0.22 (cyclohexane/EtOAc, 1:1); [
a] +33 (c 0.3,
D
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.33 (s, 1H, H-8), 8.06 (br s,
0
0
1H, NH), 7.42–7.21 (m, 20H, H arom.), 6.49 (d, 1H, J1 ,2 = 8.9 Hz,
H-10), 5.10 (d, 1H, J = 10.1 Hz, CHPh), 5.04 (d, 1H, J = 12.0 Hz, CHPh),
4.87 (dd, 1H, J5 ,6 = 2.4 Hz, J5 ,4 = 9.8 Hz, H-50), 4.78 (d, 1H,
J = 12.0 Hz, CHPh), 4.72–4.65 (m, 4H, H-40, CH2Ph, CHPh), 4.61–
0
0
0
0
4.1.11. Benzyl 1-O-acetyl-6-azido-3,4-di-O-benzyl-3-C,2-O-[(1S)-
4.58 (m, 2H, H-20, CHPh), 4.55 (d, 1H, J6 ,5 = 2.4 Hz, H-60), 4.48 (d,
0
0
1-(benzyloxy)ethylene]-6-deoxy-L-glycero–D-gluco-hepto-
1H, J = 10.8 Hz, CHPh), 4.39 (d, 1H, J8 ,9 a = 3.2 Hz, H-80), 4.32 (dd,
0
0
pyranuronate (13b)
1H, J9 a,8 = 3.2 Hz, J9 a,9 b = 9.5 Hz, H-90a), 4.09 (d, 1H, J9 b,9 a = 9.5 Hz,
H-90b), 2.59 (s, 3H, NHAc); 13C NMR (CDCl3, 100 MHz): d 171.80,
166.61 (2 ꢁ C@O), 162.99 (C-4), 152.47 (C-2 or C-6), 146.30 (C-8),
143.77 (C-2 or C-6), 137.48, 136.71, 136.61, 134.46 (4C arom.
quat.), 128.66–127.82 (20CH arom.), 118.69 (C-5), 89.72 (C-30),
84.51 (C-80), 82.76 (C-10), 77.44 (C-50), 77.37 (C-20), 74.60 (CH2Ph),
74.48 (C-90), 73.46 (CH2Ph), 72.67 (C-40), 67.80 (CH2Ph), 65.40
(CH2Ph), 62.81 (C-60), 25.20 (NHAc); HRMS (FAB) m/z: [M+Na]+
calcd for C44H41O8N835ClNa, 867.2634, and C44H41O8N837ClNa,
869.2604, found: 867.2618 and 869.2678, respectively.
0
0
0
0
0
0
To a solution of benzyl azido ester 8b (200 mg, 0.30 mmol) in
acetic anhydride (7 mL) was added dropwise sulfuric acid (21 L)
l
at 0 °C. The reaction mixture was stirred at this temperature over
30 min and then neutralized by slow addition of a NaHCO3 satu-
rated aqueous solution. The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (3 ꢁ 50 mL). The organic
layers were combined, dried (MgSO4), filtered and concentrated.
Purification by flash column chromatography (cyclohexane/
CH2Cl2/EtOAc, 10:1:1) afforded the glycosyl donor 13b (131 mg,
63%) as a colorless oil. Rf 0.50 (cyclohexane/CH2Cl2/EtOAc, 6:1:1);
[a]
+30.8 (c 0.2, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.36–7.14
D
(m, 20H, H arom.), 5.96 (d, 1H, J1,2 = 7.8 Hz, H-1), 5.28 (d, 1H,
J = 12.1 Hz, CHPh), 5.08 (d, 1H, J = 12.1 Hz, CHPh), 4.94 (d, 1H,
J = 11.1 Hz, CHPh), 4.73 (dd, 1H, J5,6 = 2.5 Hz, J5,4 = 9.9 Hz, H-5),
4.67 (d, 1H, J = 11.1 Hz, CHPh), 4.64 (d, 1H, J = 11.1 Hz, CHPh),
4.62 (d, 1H, J = 11.1 Hz, CHPh), 4.56 (s 2H, CH2Ph), 4.30 (d, 1H,
J4,5 = 9.9 Hz, H-4), 4.25 (d, 1H, J2,1 = 7.8 Hz, H-2), 4.23–4.21 (m,
2H, H-6, H-8), 4.18 (dd, 1H, J9a,8 = 3.3 Hz, J9a,9b = 9.4 Hz, H-9a),
4.03 (d, 1H, J9b,9a = 9.4 Hz, H-9b), 2.10 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 169.09, 169.02 (2 ꢁ C@O), 137.79, 137.36, 137.14,
135.06 (4C arom. quat.), 128.55–127.80 (20CH arom.), 93.63 (C-
1), 89.96 (C-3), 84.33 (C-8), 77.65 (C-2), 75.19 (C-5), 74.74 (CH2Ph),
74.09 (C-4), 73.53 (C-9), 72.82 (CH2Ph), 67.96 (CH2Ph), 65.03
(CH2Ph), 62.28 (C-6), 21.01 (OAc); HRMS (CI) m/z: [M+NH4]+ calcd
for C39H43O9N4, 711.3030, found: 711.3021.
4.1.13. 2-(Acetylamino)-6-chloro-7-(benzyl {6-azido-3,4-di-O-
benzyl-3-C,2-O-[(1S)-1-(benzyloxy)ethylene]-6-deoxy-
glycero– -gluco-heptopyranosyl}urinate)purine (14b)
Method A: BSA (65 L, 259.5 mol) was added to a suspension
the nucleobase 2-acetamido-6-chloropurine (28 mg,
mol) in anhydrous CH3CN (1 mL) under argon. The mixture
L-
D
l
l
of
129.8
l
was heated to 60 °C for 45 min to afford the crude silylated base. To
this solution was added the glycosyl donor 6R 13b (60 mg,
86.5
l
mol) dissolved in anhydrous CH3CN (1 mL) followed by slow
L, 692 mol) under argon. The solution
addition of TMSOTf (125
l
l
was stirred at 65 °C for 90 min. The mixture was cooled to room
temperature, CH2Cl2 (20 mL) was added and the organic layer
was washed with NaHCO3 saturated aqueous solution
(2 ꢁ 10 mL). The aqueous layer was extracted with CH2Cl2
(3 ꢁ 30 mL), the organic layers were combined, dried (MgSO4), fil-
tered and concentrated under reduced pressure. Purification by
flash column chromatography (cyclohexane/EtOAc, 1:1) afforded
the N7 nucleoside 6R 14b (44 mg, 58%) as a colorless oil. Method
4.1.12. 2-(Acetylamino)-6-chloro-7-(benzyl {6-azido-3,4-di-O-
benzyl-3-C,2-O-[(1S)-1-(benzyloxy)ethylene]-6-deoxy-
glycero– -gluco-heptopyranosyl}urinate)purine (14a)
Method A: BSA (54 L, 216.3 mol) was added to a suspension
the nucleobase 2-acetamido-6-chloropurine (23 mg,
mol) in anhydrous CH3CN (1 mL) under argon. The mixture
D-
D
l
l
B: BSA (32
lL, 129.6
lmol) was added to a suspension of the nucle-
of
obase 2-acetamido-6-chloropurine (14 mg, 64.8
lmol) in anhy-
108.2
l
drous CH3CN (1 mL) under argon. The mixture was heated to
was heated to 60 °C for 45 min to afford the crude silylated base. To
this solution was added the glycosyl donor 6S 13a (50 mg,
60 °C for 45 min to afford the crude silylated base. To this solution
was added the glycosyl donor 6R 13b (30 mg, 43.2
in anhydrous CH3CN (1 mL) followed by slow addition of TMSOTf
(156 L, 864 mol) under argon. The solution was stirred at 65 °C
lmol) dissolved
72.1
lmol) dissolved in anhydrous CH3CN (1 mL) followed by slow
addition of TMSOTf (104
l
L, 576.8 mol) under argon. The solution
l
l
l
was stirred at 65 °C for 90 min. The mixture was cooled to room
temperature, CH2Cl2 (20 mL) was added and the organic layer
was washed with NaHCO3 saturated aqueous solution
(2 ꢁ 10 mL). The aqueous layer was extracted with CH2Cl2
(3 ꢁ 30 mL), the organic layers were combined, dried (MgSO4), fil-
tered and concentrated under reduced pressure. Purification by
flash column chromatography (cyclohexane/EtOAc, 1:1) afforded
the N7 nucleoside 6S 14a (39 mg, 65%) as a colorless oil. Method
for 90 min. The mixture was cooled to room temperature, CH2Cl2
(20 mL) was added and the organic layer was washed with NaHCO3
saturated aqueous solution (2 ꢁ 10 mL). The aqueous layer was ex-
tracted with CH2Cl2 (3 ꢁ 30 mL), the organic layers were com-
bined, dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (cyclohex-
ane/EtOAc, 1:1) afforded the N7 nucleoside 6R 14b (20 mg, 55%) as
a colorless oil. Rf 0.31 (cyclohexane/EtOAc, 1:1); [
a] +51.3 (c 0.2,
D
B: BSA (32
l
L, 129.6
l
mol) was added to a suspension of the nucle-
mol) in anhy-
CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.31 (s, 1H, H-8), 8.30 (br s,
0
0
obase 2-acetamido-6-chloropurine (14 mg, 64.8
l
1H, NH), 7.44–7.08 (m, 20H, H arom.), 6.35 (d, 1H, J1 ,2 = 8.8 Hz,
drous CH3CN (1 mL) under argon. The mixture was heated to
H-10), 5.22 (d, 1H, J = 12.0 Hz, CHPh), 5.09 (d, 1H, J = 11.2 Hz, CHPh),
0
0
60 °C for 45 min to afford the crude silylated base. To this solution
5.05 (d, 1H, J = 12.0 Hz, CHPh), 4.83 (dd, 1H, J5 ,6 = 2.2 Hz,

F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
5115
J5 ,4 = 9.8 Hz, H-50), 4.77 (d, 1H, J = 11.2 Hz, CHPh), 4.75 (s, 2H,
CH2Ph), 4.70 (d, 1H, J = 10.4 Hz, CHPh), 4.61 (d, 1H, J = 10.4 Hz,
final concentrations of chemicals in the test were as follows:
[AChE] = 0.03 U/mL, [BChE]=0.01 U/mL, [compound] = 0.01–
100 g/mL, [DTNB] = 0.0002273 M, [ATchI] = [BTchI] = 0.0005 M.
0
0
CHPh), 4.51 (d, 1H, J4 5 = 9.8 Hz, H-4), 4.44–4.42 (m, 2H, H-20, H-
l
0
0
80), 4.31 (dd, 1H, J9 a,8 = 3.3 Hz, J9 a,9 b = 9.5 Hz, H-90a), 4.22–4.19
(m, 2H, H-60, H-90b), 2.65 (s, 3H, NHAc); 13C NMR (CDCl3,
100 MHz): d 171.61, 167.63 (2 ꢁ C@O), 162.84 (C-4), 152.36 (C-2
or C-6), 145.90 (C-8), 143.62 (C-2 or C-6), 137.48, 136.84, 136.65,
134.35 (4C arom. quat.), 128.69–127.27 (20CH arom.), 118.69 (C-
5), 89.38 (C-30), 84.42 (C-80), 82.78 (C-10), 78.96 (C-20), 78.14 (C-
50), 74.96 (CH2Ph), 74.19 (C-90), 73.85 (C-40), 73.60 (CH2Ph), 67.66
(CH2Ph), 65.42 (CH2Ph), 61.02 (C-60), 25.19 (NHAc); HRMS (FAB)
m/z: [M+Na]+ calcd for C44H41O8N835ClNa, 867.2634, and
C44H41O8N837ClNa, 869.2604, found: 867.2642 and 869.2612,
respectively.
0
0
0
0
4.3. Preliminary acute toxicity screening
Acute cytotoxicity measurements were performed by the MTT
method.29,30 The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay was used to quantify metabolically
viable cells in all samples. Adherent cells (A549 human lung fibro-
blasts) were seeded onto 96-well plates, allowed to attach for 24 h
and exposed to the test compound for the following 24 h. Positive
control (hydrogen peroxide) and negative control (pure solvent)
were also included. At 48 h of culture MTT was added to the cells
at a final concentration of 0.5 mg/mL, followed by an incubation
period of 3 h to allow the formazan crystals to form. After incuba-
tion, medium was removed, cells were washed twice to remove
4.2. Enzymatic studies
4.2.1. Spectrophotometer and chemicals
traces of medium and un-metabolized MTT, and DMSO (100 lL)
A double beam spectrophotometer ShimadzuÒ equipped with
thermostatic cell holders was used on visible range and operated
on the kinetic mode. The absorbance data were acquired in a com-
puter by means of UV Probe software. Appropriate disposable plas-
tic cuvettes PlastibrandÒ were used in the kinetic experiments. The
following materials were purchased from Aldrich: enzymes acetyl-
cholinesterase (AChE) from bovine erythrocytes and butyrylcholin-
esterase (BChE) from human serum, substrates acetylthiocholine
iodide (ATchI) and S-butyrylthiocholine iodide (BTchI) and 5,50-
dithiobis-2-nitrobenzoic acid (DTNB). Other Aldrich reagents used
for the preparation of buffers and solutions were KH2PO4, KOH and
NaHCO3. Deionized water was used to prepare the buffer pH 8.0,
the substrate and DTNB solutions.
was added to each well. Solubilization of formazan crystals was
performed by agitation in a 96-well plate shaker for 20 min at
room temperature. Absorbance of each well was quantified at
550 nm using 620 nm as reference wavelength on a scanning mul-
tiwell spectrophotometer (automated plate reader).
Acknowledgments
This work was supported by Fundação para Ciência e Tecnologia
(FCT, Project POCI/QUI/59672/2004, Portugal). Filipa Marcelo
thanks FCT for the PhD research grant (SFRH/BD/17775/2004). Fili-
pa V. M. Silva and Margarida Goulart thank FCT for the Post-Doc
fellowships SFRH/BPD/14408/2003 and SFRH/BPD/20902/2004,
respectively. Dr. Karl Heinz Hellwich is also gratefully acknowl-
edged for his suggestions for the IUPAC names of some
compounds.
4.2.2. Solutions preparation
Preparation of 0.1 M phosphate buffer pH 8.0: KH2PO4
(136.1 mg) was dissolved in water (10 mL) and adjusted with
KOH to a pH of 8.0 0.1. Buffer was freshly prepared and stored
in the refrigerator.
References and notes
AChE solution 1.32 U/mL: the enzyme (1.02041U, 10 lL, 4.4 mg)
was dissolved in freshly prepared buffer pH 8.0 (1.0 mL).
BChE solution 0.44 U/mL: The enzyme (2.9762 U, 1.0 mg) was
dissolved in freshly prepared buffer pH 8.0 (6.764 mL).
DTNB solution 0.01 M: DTNB (3.96 mg) was dissolved in water
(1 mL) containing sodium hydrogen carbonate (1.5 mg).
ATchI solution 0.022 M: ATchI (6.4 mg) was dissolved in water
(1 mL).
1. The Biology of Cholinesterases; Silver, A., Ed.; Elsevier: New York, 1974.
2. Whitehouse, P. J.; Price, D. L.; Clark, A. W.; Coyle, J. T.; DeLong, M. R. Ann. Neurol.
1981, 10, 122.
3. Bartus, R. T.; Dean, R. L. I.; Beer, B.; Lippa, A. S. Science 1982, 217, 408.
4. Coyle, J. T.; Price, D. L.; DeLong, M. R. Science 1983, 219, 1184.
5. Guttman, R.; Altman, R. D.; Nielsen, N. H. Arch. Fam. Med. 1999, 8, 347.
6. Winkler, J.; Thal, J. L.; Gage, F. H.; Fisher, J. L. J. Mol. Med. 1998, 76, 555.
7. Darvesh, S.; Hopkins, D. A.; Geula, C. Nat. Rev. Neurosci. 2003, 4, 131.
8. Darvesh, S.; MacKnight, C.; Rockwood, K. Int. Psychogeriatr. 2001, 13, 461.
9. Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.; Scali, C.; Yu, Q. S.;
Mamczarz, J.; Holloway, H. W.; Giordano, T.; Chen, D.; Furukawa, K.;
Sambamurti, K.; Brossi, A.; Lahiri, D. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
17213.
10. Kurz, A. J. Neural. Trans. Suppl. 1998, 54, 295.
11. Jann, M. W. Pharmacotherapy 2000, 20, 1.
12. Sugimoto, H. Pure Appl. Chem. 1999, 71, 2031.
13. Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y. Curr. Med. Chem. 2000, 7,
303.
BTchI solution 0.022 M: BTchI (7.0 mg) was dissolved in water
(1 mL).
All solutions were stored in eppendorf caps (100 lL aliquots) in
the refrigerator. The pure compounds and the control (rivastig-
mine) were initially dissolved in DMSO and diluted with distilled
water until concentrations decrease to values between 4.4 mg/
mL and 0.00044 mg/mL, to yield the final concentrations for the
enzymatic test between 100 lg/mL and 0.01 lg/mL. No inhibition
was detected by residual DMSO (<0.5%) at the reaction cuvette.
14. Zarotsky, V.; Sramek, J. J.; Cutler, N. R. Am. J. Health Syst. Pharm. 2003, 60, 446.
15. Darvesh, S.; Walsh, R.; Kumar, R.; Caines, A.; Roberts, S.; Magee, D.; Rockwood,
K.; Martin, E. Alzheimer Dis. Assoc. Disord. 2003, 17, 117.
16. Greig, N. H.; Lahiri, D. K.; Sambamurti, K. Int. Psychogeriatr. 2002, 14, 77.
17. Marcelo, F.; Jiménez-Barbero, J.; Marrot, J.; Rauter, A. P.; Sinaÿ, P.; Blériot, Y.
Chem. Eur. J. 2008, 14, 10066.
18. Marcelo, F.; Abou-Jneid, R.; Sollogoub, M.; Marrot, J.; Rauter, A. P.; Blériot, Y.
Synlett 2009, 1269.
19. Fairbanks, A. J.; Sinaÿ, P. Synlett 1995, 277.
20. Czernecki, S.; Horns, S.; Valéry, J.-M. J. Org. Chem. 1995, 60, 650.
21. Garner, P.; Yoo, J. U.; Saraku, R. Tetrahedron 1992, 48, 4259.
22. Hori, H.; Nishida, Y.; Ohrui, H.; Meguro, H. J. Org. Chem. 1989, 54, 1346.
23. Ellman, G. L.; Courtney, D. K.; Andres, V.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
24. Mendelson, I.; Kronman, C.; Ariel, N.; Shafferman, A.; Velan, B. Biochem. J. 1998,
334, 251.
25. Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J. C.; Nachon, F. J. Biol.
Chem. 2003, 278, 41141.
4.2.3. AChE and BChE activity assay
A mixture of the buffer (200
and new synthetic compounds (5
kept for 15 min at 30 °C in a heated water bath, and then the sub-
strate reagent (5 L) was added to start the enzymatic reaction.
l
L), enzyme (5
lL), DTNB (5 lL)
lL) solutions was prepared and
l
The absorbance data along reaction time was taken for 4 min under
a controlled temperature of 30 °C. At least four replicates were
made. Several blank assays without the new synthetic compounds
were carried out in order to determine the average Vmax. Also as-
says without the enzyme and the inhibitor compound were carried
out to check for any non-enzymatic hydrolysis of the substrate. The

5116
F. Marcelo et al. / Bioorg. Med. Chem. 17 (2009) 5106–5116
26. Saxena, A.; Redman, A. M.; Jiang, X.; Lockridge, O.; Doctor, B. P. Biochemistry
1997, 36, 14642.
27. Meier, C.; Ducho, C.; Görbig, U.;Esnouf, R.; Balzarini, J. J. Med. Chem. 2004, 47, 2839.
28. Darvesh, S.; McDonald, R. S.; Penwell, A.; Conrad, S.; Darvesh, K. V.; Mataija, D.;
Gomez, G.; Caines, A.; Walsh, R.; Martin, E. Bioorg. Med. Chem. 2005, 13, 211.
29. Darvesh, S.; McDonald, R. S.; Darvesh, K. V.; Mataija, D.; Conrad, S.; Gomez, G.;
Walsh, R.; Martin, E. Bioorg. Med. Chem. 2007, 15, 6367.
30. Freshney, R. L. Culture of Animal Cells: A Manual of Basic Techniques; Wiley-Liss:
New York, 2000.
31. Plumb, J. A.; Milroy, R.; Kaye, S. B. Cancer Res. 1989, 49, 4435.