Microwave-assisted synthesis of novel purine nucleosides as selective cholinesterase inhibitors

Article abstract of DOI:10.1039/c4ob00142g

Alzheimer's disease (AD), the most common form of senile dementia, is characterized by high butyrylcholinesterase (BChE) levels in the brain in later AD stages, for which no treatment is available. Pursuing our studies on selective BChE inhibitors, that may contribute to understand the role of this enzyme in disease progression, we present now microwave-assisted synthesis and anticholinesterase activity of a new nucleoside series embodying 6-chloropurine or 2-acetamido-6-chloropurine linked to d-glucosyl, d-galactosyl and d-mannosyl residues. It was designed to assess the contribution of sugar stereochemistry, purine structure and linkage to the sugar for cholinesterase inhibition efficiency and selectivity. Compounds were subjected to Ellman's assay and their inhibition constants determined. The α-anomers were the most active compounds, while selectivity for BChE or acetylcholinesterase (AChE) inhibition could be tuned by the purine base, by the glycosyl moiety and by N ⁷-ligation. Some of the nucleosides were far more potent than the drug galantamine, and the most promising competitive and selective BChE inhibitor, the N⁷-linked 2-acetamido-α-d-mannosylpurine, showed a K_i of 50 nM and a selectivity factor of 340 fold for BChE over AChE. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00142g

Organic &
Biomolecular Chemistry
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Microwave-assisted synthesis of novel purine
nucleosides as selective cholinesterase inhibitors†
S. Schwarz,a,b R. Csukb and A. P. Rauter*a
Cite this: Org. Biomol. Chem., 2014,
12, 2446
Alzheimer’s disease (AD), the most common form of senile dementia, is characterized by high butyrylcholi-
nesterase (BChE) levels in the brain in later AD stages, for which no treatment is available. Pursuing our
studies on selective BChE inhibitors, that may contribute to understand the role of this enzyme in disease
progression, we present now microwave-assisted synthesis and anticholinesterase activity of a new nucleo-
side series embodying 6-chloropurine or 2-acetamido-6-chloropurine linked to D-glucosyl, D-galactosyl and
D-mannosyl residues. It was designed to assess the contribution of sugar stereochemistry, purine structure
and linkage to the sugar for cholinesterase inhibition efficiency and selectivity. Compounds were subjected
to Ellman’s assay and their inhibition constants determined. The α-anomers were the most active com-
pounds, while selectivity for BChE or acetylcholinesterase (AChE) inhibition could be tuned by the purine
base, by the glycosyl moiety and by N7-ligation. Some of the nucleosides were far more potent than the
drug galantamine, and the most promising competitive and selective BChE inhibitor, the N7-linked 2-acet-
amido-α-D-mannosylpurine, showed a Ki of 50 nM and a selectivity factor of 340 fold for BChE over AChE.
Received 19th January 2014,
Accepted 7th February 2014
DOI: 10.1039/c4ob00142g
www.rsc.org/obc
on the AChE inhibitors galantamine12 and donepezil13,14 and
the dual cholinesterase inhibitor rivastigmine15 that raise the
Introduction
More than 100 years after Alois Alzheimer first described a ACh level and improve cognitive abilities.16,17 However these
new neurodegenerative disease,1 that now bears his name, drugs are only effective in the early stages of disease.
there is still no cure available for it. Alzheimer’s disease (AD)
Histochemical analysis of Alzheimer’s disease (AD) brain
is a multifactorial disease characterized by the presence of tissues indicates that BChE is also present in β-amyloid (Aβ)
β-amyloid plaques and neurofibrillar tangles in the brain and plaques and recent studies in a transgenic mouse model of AD
also a decrease of the level of the neurotransmitter acetyl- have suggested that it may play a role in AD plaque matu-
choline (ACh), that causes a dysfunction of the cholinergic ration.18 Currently under investigation,19 the influence of
activity, which results in
a
decline in memory and BChE on AD pathology is still unclear.
Selective BChE inhibitors will facilitate a better understand-
recognition.2–4 The level of acetylcholine in the brain is con-
trolled by the hydrolase acetylcholinesterase (AChE, E.C. ing of the role of this enzyme on AD progression. One of the
3.1.1.7). Also butyrylcholinesterase (BChE, E.C. 3.1.1.8) is able first successful attempts to obtain selective BChE inhibitors
to hydrolyze ACh, although it does not have the same affinity was accomplished by Greig et al. investigating derivatives of
as AChE.5 It is known that about 80% of the cholinergic cymserin,7 followed by Takahashi describing norcymserin
activity inside the human brain is caused by AChE.6 However derivatives.20 Other groups succeeded by dealing with deriva-
AChE levels decrease in AD, while those of BChE are highly tives of polyphenol21 and quinazolinimine.22 The use of
increased. The ratio of BChE/AChE changes from 0.5 in the purine nucleotides appeared to be promising and cycloSal pro-
normal brain to 11 in the AD brain7 but the reduced AChE nucleotides showed selectivity towards inhibition of BChE.23,24
activity may be upregulated by BChE,8,9 whose activity is More recently Marcelo et al. synthesized a series of highly
doubled in advanced AD.10,11 Current treatment of AD is based active purine nucleosides.25 Those compounds, bearing a
benzyl protected bicyclic sugar moiety N7 and β-linked to
2-acetamido-6-chloropurine, exhibited BChE selectivity, and
aCentro de Química e Bioquímica/Departamento de Química e Bioquímica,
Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Ed. C8, Piso 5,
1749-016 Lisboa, Portugal. E-mail: aprauter@fc.ul.pt
one of them showed nM inhibition of the same order of mag-
nitude as that of rivastigmine. These results encouraged us to
launch our investigation into the impact of the sugar and
purine structures on this new family of selective BChE inhibi-
tors. Therefore, a series of benzylated glycosyl donors with
D-gluco, D-manno and D-galacto configuration were linked either
bBereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-
Mothes-Str. 2, D-06120 Halle (Saale), Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00142g
2446 | Org. Biomol. Chem., 2014, 12, 2446–2456
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to 6-chloropurine (CP) or 2-acetamido-6-chloropurine (ACP). the concentration of the activator. In ACP glucosylation the
The reaction conditions of Marcelo et al.25 were optimized for same trends were observed but N9-selectivity detected for CP
this family of compounds and microwave-assisted reactions glucosylation did not occur.
were carried out for the glycosylation reaction. The inhibitory
When using microwave irradiation instead of conventional
activities of all compounds were determined by Ellman’s assays heating, the time taken for glucosylation completion of CP and
using AChE and BChE, aiming to find efficient and selective ACP dropped significantly from 2 h to 15 min but reaction
BChE inhibitors of a simplified structure when compared to the regioselectivity and stereoselectivity were not significantly
parent nucleosides previously described by our group.25
altered. Consequently, all compounds were further synthesized
by microwave-assisted synthesis (Scheme 1).
The anomeric configuration as well as the substitution at
N7 or N9 of the compounds was determined from their 1H-
and 13C NMR spectra, COSY, HMQC, HMBC and gHSQC
experiments. The chemical shift of purine carbon 5 can be
used to distinguish between N7- and N9-substitution. It was
found to be about δ = 122 ppm for N7 CP and about δ =
118 ppm for N7 ACP, while substitution at N9 resulted in
chemical shifts of about δ = 131 ppm for CP and about δ =
128 ppm for ACP derivatives. The anomeric configuration was
Results and discussion
Chemistry
6-Chloropurine nucleoside synthesis has been described start-
ing from 1-O-acetyl glycosyl donors and mediated by stannic
chloride in acetonitrile to give N9-regioselectivity with the ribo-
furanosyl donor26 and N7-regioselectivity with the glucosyl
donor.26 Alternatively, TMSOTf mediated reaction gave the
product resulting from thermodynamic control, the N9-regio-
isomer.27,28 Furthermore, the direct coupling of a persilylated
purine with a methyl furanoside using Lewis acid activation of
the anomeric centre by stannic chloride30 or by TMSOTf31 has
been accomplished. Also a furanosyl halide has been activated
by stannic chloride to give a purine nucleoside.29 Other
leaving groups have also been used, namely the phenylsulfanyl
group activated by N-iodosuccimide/triflic acid to yield the N9-
regioisomer.27 Marcelo et al. applied TMSOTf in acetonitrile
for the first time to couple methyl bicyclic hexosides to a
purine scaffold with β-stereoselectivity and N7-regioselectivity
in 58–65% yield.25 Hence, we started from the perbenzylated
methyl glycoside precursors and investigated their conjugation
with persilylated CP as well as ACP. The influence of the con-
centration of activating TMSOTf as well as that of microwave
irradiation onto yield and reaction stereo- and regioselectivity
were studied. Four products were formed that differed in their
anomeric configuration and linkage of the glycosyl group to
purine either in the N7- or N9-position (Table 1). For CP gluco-
sylation the ratio α/β is in favor of the β-anomer for both regio-
isomers, but the β-selectivity for the N7-isomer increased with
3
unambiguously assigned from the coupling constant J1,2 and
the chemical shift of H-1 of the glycosyl group. For D-gluco or
3
D-galacto configurated products the coupling constants J1,2
=
5 Hz (for glucosyl group) and 3J1,2 = 3 Hz (for galactosyl group)
confirm the α-configuration with H-1 chemical shifts observed
between δ = 6.1 and δ = 6.4 ppm. The corresponding
β-anomers presented H-1 chemical shifts between δ = 5.4 and
3
δ = 5.7 ppm and coupling constants J1,2 ranging from 7 to
9 Hz, characteristic of the trans-diaxial position of H-1 and
Table 1 Conditions for the reaction of methyl 2,3,4,6-tetra-O-benzyl-
α-D-glucoside with the purines CP or ACPa
TMSOTf
Purine (eq.)
Total
Time
N7/N9 N7 α/β N9 α/β yield
CP
CP
CP
CP
ACP
ACP
ACP
ACP
2
4
8
8
2
4
8
8
2 h
2 h
2 h
1/1.4
1/3.8
1/3.4
1/1.7
1/2.6
1/3.6
1/2.2
1.1/1
1/2.2
1/6.2
1/5.0
1/6.0
1/5.8
1/5.9
1/4.6
1/4.0
1/8.5
1/8.2
1/9.3
56%
75%
78%
81%
2%
80%
71%
74%
15 min (mw) 1/2.5
2 h
2 h
2 h
1/1.4
1.3/1
1/1.2
15 min (mw) 1.3/1
a Yields and product ratios were determined by 1H NMR experiments;
1 equivalent of the monosaccharide and 1.5 equivalents of the
silylated purine in dry acetonitrile were used under conventional
heating or microwave-assisted heating (mw) at 65 °C. No product was
formed with both purines in the presence of 1 equivalent of TMSOTf.
Scheme 1 Reagents and conditions: silylated CP or ACP, TMSOTf,
acetonitrile, microwave irradiation (150 W), 65 °C, 15 min.
This journal is © The Royal Society of Chemistry 2014
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H-2. For D-manno configurated compounds, coupling constants
3J1,2 of approx. J = 9 Hz for the α-anomers and 1 Hz for the
β-anomers were obtained.
Table 2 Inhibitory constant Ki for nucleosides 1–24 as determined by
Ellman’s assay with BChE and AChE in comparison to galantamine
hydrobromidea
In the 13C NMR spectra, chemical shifts of δ = 79 to δ =
81 ppm for the α-anomers and of δ = 83 to δ = 85 ppm for the
β-anomers were found in excellent agreement with those pre-
viously reported for related nucleosides.25
Purine N7/N9 α/β Sacch Ki (µM) (BChE) Ki (µM) (AChE)
Galantamine hydrobromide
6-Chloropurine (CP)
2-Acetamido-6-chloropurine (ACP)
9.4 0.7
>100
>100
>100
>100
>20
>2
>100
>100
>20
>10
>100
30.9 2.2
9.6 0.7
2.8 0.3
>20
2.5 0.3
>100
0.5 0.0
55.5 3.9c
26.1 2.8d
28.9 2.1
17.5 2.6e
9.6 2.3
>2
17.0 2.0
16.0 2.7f
>20
5.6 0.8
25.6 2.1
23.4 3.6
28.5 6.5
2.4 0.3
>20
The highest β/α ratio (9 : 1), determined by 1H NMR, was
found in the glucosylation of 2-acetamido-6-chloropurine, while
galactosylation of 6-chloropurine afforded the highest β/α ratio
(8 : 1). N9-regioselectivity was observed for the chloropurine
nucleosides bearing galactosyl or glucosyl residues while intro-
duction of the substituent 2-acetamido favours N7-linked nucleo-
sides leading to a N9/N7 ratio ca. 1.0. However, the N7-linked
D-manno nucleosides bearing the 2-acetamido-6-chloropurine
exhibit a β/α ratio (3.5 : 1) while for the 6-chloropurine no selecti-
vity occurs. With both the purine bases, α-selectivity is found for
the N9-linked nucleosides.
1
2
3
4
5
6
7
8
9
CP
CP
CP
CP
CP
CP
CP
CP
CP
N7
N9
N7
N7
N9
N9
N7
N9
N9
N7
N7
N9
N9
N7
N7
N9
N7
N7
N9
N9
N7
N7
N9
N9
β
β
α
β
α
β
β
α
β
α
β
α
β
α
β
β
α
β
α
β
α
β
α
β
Glc
Glc
Glc
Glc
Glc
Glc
Gal
Gal
Gal
10 CP
11 CP
12 CP
13 CP
Man
Man
Man
Man
Glc
Glc
Glc
Gal
Gal
Gal
Gal
14 ACP
15 ACP
16 ACP
17 ACP
18 ACP
19 ACP
20 ACP
21 ACP
22 ACP
23 ACP
24 ACP
>10
23.0 1.9
>2
42.0 9.2
>20
>2
>2
Biology
50.0 7.0
>20
>2
All compounds were subjected to Ellman’s assay32 varying sub-
strate and compound concentration to evaluate the inhibition
constants Ki (dissociation constant of enzyme–inhibitor
>2
>2
Man
Man
Man
Man
0.05 0.01b
1.4 0.1
10.3 2.6
>10
18.1 4.8
3.0 0.3
17.1 2.5
>10
complex in competitive inhibition) and K′ (dissociation con-
i
stant of enzyme–substrate–inhibitor complex in non-competi-
tive inhibition) and the type of inhibition caused by the 24
synthesized nucleosides. The data for the compounds, for
their parent purines and for galantamine hydrobromide – a
common drug used for treating AD – are compiled in Table 2.
Since the glycosyl residues carry four benzyl groups, with the
exception of compounds 1 and 2, the solubility was poor
under the assay conditions. Thus, for some of the compounds,
specific data could not be obtained. For these compounds
the maximum compound concentration was too low to cause
a detectable inhibition, although the cut-off of our assay
(100 µM) was not reached. Hence, it was not possible to receive
enough data for a definite statement of structure–activity
relationship studies for all the compounds tested. Neverthe-
less, some structural principles as well as tendencies could be
determined. First of all, most of the compounds showed a
competitive inhibition on both cholinesterases as galantamine
hydrobromide did. Galantamine hydrobromide has shown a
higher impact on AChE than on BChE. The selectivity factor of
galantamine, given by the quotient of Ki (AChE) and Ki (BChE),
is 0.06. The unsubstituted purines CP and ACP showed no
inhibition on BChE at all, but a linear mixed type inhibition
on AChE. This allowed the determination of their selectivity
for AChE inhibition. The D-gluco and D-galacto chloropurine
nucleosides were selective for AChE inhibition, with the exclu-
sion of both the perbenzylated N7-β-nucleosides that could not
be evaluated due to their poor solubility under the assay con-
ditions. In addition, N7- or N9-ligation does not seem to be
crucial for AChE selective inhibition of chloropurine nucleo-
sides, also including those of the D-manno series.
a Four different substrate concentrations and 4 different inhibitor
concentrations were used. Each experiment was performed in
triplicate. Poor compound solubility limited the evaluation of Ki when
lying above the given concentration. b Ki′ = 0.7 0.1 µM. c Ki′ = 115.4
20.5 µM. d Ki′ = 85.8
10.7 µM. e Ki′ = 74.3
7.1 µM. f Ki′ = 44.6
14.3 µM.
exception of both N9-β-diastereomers, inhibitory constants
ranging from 50 nM to 30 µM were determined. It is note-
worthy that low inhibition constants were shown by both
anomers N7-linked to ACP when compared to their N7-linked
CP counterparts. The α-anomers were in general the most
active compounds for the inhibition of both cholinesterases.
However, selectivity for BChE inhibition was mostly shown by
the 2-acetamidochloropurine nucleosides, particularly those
which exhibit N7-ligation to the α-D-glucosyl or the α-D-manno-
syl group, with the exception of compound 11, the single
chloropurine nucleoside that is a BChE selective inhibitor.
Within all 24 compounds studied, compound 21, bearing a
2-acetamido-6-chloropurine base N7-linked to a α-D-mannosyl
group, was the most potent BChE inhibitor possessing a Ki of
50 nM. Furthermore, this nucleoside showed an extraordinary
selectivity as demonstrated by a selectivity factor of 340.
Experimental
General experimental procedures
Reagents were purchased from commercial suppliers without
The introduction of the perbenzylated mannosyl substitu- any further purification. Microwave assisted synthesis was per-
ent increased the inhibitory activity considerably. With the formed with a CEM Discover and Explorer SP. Melting points
2448 | Org. Biomol. Chem., 2014, 12, 2446–2456
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were measured with a Melting Point Apparatus, SMP3, Stuart 5.48 (dd, J = 9.4 Hz, J = 9.4 Hz, 1H, H-3′), 5.31 (dd, J = 9.6 Hz,
Scientific, Bibby and were not corrected. NMR spectra were J = 9.4 Hz, 1H, H-4′), 4.30 (dd, J = 12.7 Hz, J = 4.7 Hz, 1H, H-6′
recorded on a BRUKER Avance 400 spectrometer at 298 K with a), 4.16 (d, J = 12.7 Hz, 1H, H-6′b), 4.05 (m, 1H, H-5′), 2.08 (s,
trimethylsilane as an internal standard, δ are given in ppm 3H, Ac-CH3), 2.07 (s, 3H, Ac-CH3), 2.04 (s, 3H, Ac-CH3), 1.78 (s,
and J in Hz. Mass spectra were taken on a FINNIGAN MAT TSQ 3H, Ac-CH3). 13C NMR (100 MHz, CDCl3): δ 170.4 (Ac-COO),
7000 (electrospray, voltage 4.5 kV, sheath gas nitrogen) instru- 169.9 (Ac-COO), 169.3 (Ac-COO), 168.9 (Ac-COO), 152.5 (C-2),
ment. Elemental analyses were carried out on a Foss-Heraeus 151.7 (C-4), 151.6 (C-6), 142.9 (C-8), 131.5 (C-5), 80.8 (C-1′),
Vario EL unit. Optical rotations were determined on a Perkin- 75.2 (C-5′), 72.7 (C-3′), 70.1 (C-2′), 67.6 (C-4′), 61.4 (C-6′), 20.7
Elmer 341 polarimeter. TLC was performed on silica gel (Ac-CH3), 20.5 (Ac-CH3), 20.5 (Ac-CH3), 20.1 (Ac-CH3). Elemen-
(Merck 5554). Solvents were dried before use according to tal anal. calcd for C19H21ClN4O9: C, 47.07; H, 4.37; N, 11.56;
usual procedures. The purity of the compounds was checked found: C, 46.93; H, 4.47; N, 11.47.
by HPLC-DAD and found to be >95% for each compound.
6-Chloro-7-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)purine
(3), 6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine
(4), 6-chloro-9-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)purine
(5) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-
purine (6). The compounds were obtained by the reaction of
methyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (100 mg,
0.18 mmol) and CP (44 mg, 0.27 mmol) according to the
general procedure. Purification by column chromatography
(EtOAc–cyclohexane 1 : 1) yielded 3 (7.3 mg; 6%), 4 (20.7 mg;
17%), 5 (12.2 mg; 10%) and 6 (54.8 mg; 45%).
N-glycosylation: general procedure
N,O-Bis(trimethylsilyl)acetamide (BSA) (1.5 eq. for CP and
3.0 eq. for ACP) was added to a mixture of the respective
purine (1.5 eq.) in dry acetonitrile. The mixture was stirred at
room temperature for 40 min.
Then, the corresponding methyl glycoside (1 eq.), dissolved
in dry acetonitrile, and trimethylsilyl trifluoromethanesulfo-
nate (TMSOTf) were added. The reaction was performed under
microwave irradiation (150 W, 65 °C, 15 min). The mixture was
poured into dichloromethane, washed with a saturated solu-
tion of Na2CO3 and extracted with dichloromethane (3 ×
15 mL). The combined organic layers were washed with brine,
dried (MgSO4) and concentrated and compounds isolated and
purified by column chromatography.
6-Chloro-7-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)purine (1)
and 6-chloro-9-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)purine
(2). The compounds obtained by the reaction of methyl
2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside (70 mg, 0.18 mmol)
and CP (44 mg, 0.27 mmol) according to the general pro-
cedure. Purification by column chromatography (EtOAc–cyclo-
hexane 1 : 1) yielded 1 (17.5 mg; 20%) and 2 (35.8 mg; 41%).
Data for compound 1. Colourless oil; Rf = 0.35 (EtOAc–
hexane, 3 : 1). [α]2D0 −12° (c 1.03, CHCl3). ESI-MS m/z (%): 485.0
([M + H]+, 100), 507.3 ([M + Na]+, 22), 969.9 ([2M + H]2+, 10),
991.0 ([2M + Na]+, 16). 1H NMR (400 MHz, CDCl3): δ 8.94 (s,
1H, H-2), 8.50 (s, 1H, H-8), 6.18 (d, J = 8.1 Hz, 1H, H-1′), 5.69
(dd, J = 8.9 Hz, J = 8.1 Hz, 1H, H-2′), 5.48 (dd, J = 9.3 Hz, J =
8.8 Hz, 1H, H-3′), 5.28 (dd, J = 9.6 Hz, J = 9.3 Hz, 1H, H-4′),
4.28 (dd, J = 12.5 Hz, J = 4.7 Hz, 1H, H-6′a), 4.18 (d, J = 12.5 Hz,
1H, H-6′b), 4.10 (m, 1H, H-5′), 2.08 (s, 6H, 2 × Ac-CH3), 2.05 (s,
3H, Ac-CH3), 2.04 (s, 3H, Ac-CH3). 13C NMR (100 MHz, CDCl3):
δ 170.4 (Ac-COO), 169.9 (Ac-COO), 169.2 (Ac-COO), 169.0 (Ac-
COO), 162.2 (C-4), 153.1 (C-2), 146.3 (C-8), 142.9 (C-6), 122.2
(C-5), 82.6 (C-1′), 75.0 (C-5′), 72.9 (C-3′), 69.8 (C-2′), 67.5 (C-4′),
61.4 (C-6′), 20.6 (Ac-CH3), 20.5 (Ac-CH3), 20.5 (Ac-CH3), 20.2
(Ac-CH3). Elemental anal. calcd for C19H21ClN4O9: C, 47.07; H,
4.37; N, 11.56; found: C, 46.86; H, 4.50; N, 11.31.
Data for compound 3. Colourless oil; Rf = 0.27 (EtOAc–hexane
1 : 2). [α]2D0 −13° (c 0.88, CHCl3). ESI-MS m/z (%): 676.9 ([M +
H]+, 100), 699.1 ([M + Na]+, 20), 1353.7 ([2M + H]+, 22), 1374.9
([2M + Na]+, 12). 1H NMR (400 MHz, CDCl3): δ 8.85 (s, 1H,
H-2), 8.70 (s, 1H, H-8), 7.43–7.02 (m, 18H, Bn H), 6.90 (m, 2H,
Bn H), 6.35 (d, J = 4.8 Hz, 1H, H-1′), 4.85 (d, J = 11.3 Hz, 1H,
Bn-CHH), 4.80 (d, J = 11.3 Hz, 1H, Bn-CHH), 4.75 (d, J = 11.1
Hz, 1H, Bn-CHH), 4.70 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.57 (d,
J = 11.1 Hz, 1H, Bn-CHH), 4.56 (d, J = 12.2 Hz, 1H, Bn-CHH),
4.48 (d, J = 12.2 Hz, 1H, Bn-CHH), 4.32 (d, J = 12.1 Hz, 1H, Bn-
CHH), 4.12 (dd, J = 6.8 Hz, J = 4.8 Hz, 1H, H-2′), 4.06 (dd, J =
6.8 Hz, J = 6.6 Hz, 1H, H-3′), 3.86 (dd, J = 9.1 Hz, J = 6.6 Hz,
1H, H-4′), 3.68–3.64 (m, 2H, H-6′a and H-5′), 3.56 (m, 1H,
H-6′b). 13C NMR (100 MHz, CDCl3): δ 161.7 (C-4), 152.4 (C-2),
148.7 (C-8), 143.0 (C-6), 137.6 (Bn-Cq), 137.5 (Bn-Cq), 137.4
(Bn-Cq), 136.1 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn),
128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.4
(Bn), 128.2 (Bn), 128.1 (Bn), 128.1 (Bn), 128.1 (Bn), 128.1 (Bn),
128.0 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7
(Bn), 121.7 (C-5), 81.0 (C-1′), 80.6 (C-3′), 77.2 (C-2′), 76.5 (C-4′),
74.4 (Bn-CH2), 74.2 (Bn-CH2), 73.9 (Bn-CH2), 73.5 (Bn-CH2),
73.2 (C-5′), 67.9 (C-6′). Elemental anal. calcd for C39H37ClN4O5:
C, 69.17; H, 5.51; N, 8.27; found: C, 68.86; H, 5.72; N, 8.12.
Data for compound 4. Colourless oil; Rf = 0.10 (EtOAc–hexane
1 : 2). [α]2D0 −9° (c 1.01, CHCl3). ESI-MS m/z (%): 587.1 ([M − Bn
+ H]+, 90), 609.3 ([M − Bn + Na]+, 48), 677.0 ([M + H]+, 100). 1H
NMR (400 MHz, CDCl3): δ 8.84 (s, 1H, H-2), 8.23 (s, 1H, H-8),
7.40–7.17 (m, 15H, BnH), 7.07–6.95 (m, 3H, BnH), 6.74 (m, 2H,
BnH), 5.72 (br, 1H, H-1′), 5.01 (d, J = 10.9 Hz, 1H, Bn-CHH),
4.95 (d, J = 10.9 Hz, 1H, Bn-CHH), 4.89 (d, J = 10.7 Hz, 1H,
Bn-CHH), 4.64 (d, J = 10.7 Hz, 1H, Bn-CHH), 4.63 (d, J =
11.5 Hz, 1H, Bn-CHH), 4.54 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.48
(d, J = 12.1 Hz, 1H, Bn-CHH), 4.23 (d, J = 11.5 Hz, 1H, Bn-
CHH), 3.97 (m, 1H, H-2′), 3.95–3.86 (m, 2H, H-3′ and H-5′),
3.78–3.70 (m, 3H, H-4′ and H-6′a and H-6′b). 13C NMR
Data for compound 2. Colourless crystals; mp 175–179 °C;
Rf = 0.69 (EtOAc–hexane, 3 : 1). [α]2D0 −15° (c 1.08, CHCl3).
ESI-MS m/z (%): 485.1 ([M + H]+, 100), 507.4 ([M + Na]+, 30),
969.8 ([2M + H]2+, 8), 991.3 ([2M + Na]+, 18). 1H NMR
(400 MHz, CDCl3): δ 8.79 (s, 1H, H-2), 8.32 (s, 1H, H-8), 5.95 (d,
J = 9.4 Hz, 1H, H-1′), 5.67 (dd, J = 9.4 Hz, J = 9.4 Hz, 1H, H-2′),
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(100 MHz, CDCl3): δ 161.4 (C-4), 152.1 (C-2), 147.0 (C-8), 143.1 calcd for C39H37ClN4O5: C, 69.17; H, 5.51; N, 8.27; found: C,
(C-6), 137.8 (Bn-Cq), 137.5 (Bn-Cq), 137.5 (Bn-Cq), 135.8 (Bn- 69.02; H, 5.38; N, 8.11.
Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn),
128.4 (Bn), 128.2 (Bn), 128.2 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (7),
6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine
6-chloro-9-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-
(Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.8 (Bn), purine (8) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyr-
127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 122.1 (C-5), 86.0 anosyl)purine (9). The compounds were obtained by the reac-
(C-3′), 85.2 (C-1′), 80.1 (C-2′), 78.1 (C-4′), 77.2 (C-5′), 75.9 (Bn- tion of methyl 2,3,4,6-tetra-O-benzyl-α-D-galactopyranoside
CH2), 75.3 (Bn-CH2), 74.7 (Bn-CH2), 73.5 (Bn-CH2), 68.2 (C-6′). (120 mg, 0.21 mmol) and CP (49 mg, 0.32 mmol) according to
Elemental anal. calcd for C39H37ClN4O5: C, 69.17; H, 5.51; the general procedure. Purification by column chromatography
N, 8.27; found: C, 68.98; H, 5.42; N, 8.02.
(EtOAc–cyclohexane 1 : 1) yielded 7 (17 mg; 12%), 8 (9.9 mg;
7%) and 9 (60.9 mg; 43%).
Data for compound 7. Colourless oil; Rf = 0.30 (EtOAc–hexane
Data for compound 5. Colourless oil; Rf = 0.55 (EtOAc–hexane
1 : 2). [α]2D0 +28° (c 1.07, CHCl3). ESI-MS m/z (%): 677.1 ([M +
H]+, 100), 699.3 ([M + Na]+, 16), 1354.8 ([2M + H]+, 18). 1H 1 : 1). [α]D20 −22° (c 1.00, CHCl3). ESI-MS m/z (%): 587.0 ([M −
NMR (400 MHz, CDCl3): δ 8.67 (s, 1H, H-2), 8.40 (s, 1H, H-8), Bn + H]+, 100), 609.3 ([M − Bn + Na]+, 70), 676.9 ([M + H]+, 7).
7.40–7.08 (m, 18H, BnH), 6.92 (m, 2H, BnH), 6.26 (d, J = 1H NMR (400 MHz, CDCl3): δ 8.83 (s, 1H, H-2), 8.26 (s, 1H,
4.9 Hz, 1H, H-1′), 4.86 (d, J = 11.4 Hz, 1H, Bn-CHH), 4.80 (d, J = H-8), 7.44–7.23 (m, 15H, BnH), 7.11–6.96 (m, 3H, BnH), 6.75
11.4 Hz, 1H, Bn-CHH), 4.74 (d, J = 11.0 Hz, 1H, Bn-CHH), 4.60 (m, 2H, BnH), 5.78 (d, J = 8.1 Hz, 1H, H-1′), 5.01 (d, J =
(d, J = 11.8 Hz, 1H, Bn-CHH), 4.54 (d, J = 11.0 Hz, 1H, Bn- 11.2 Hz, 1H, Bn-CHH), 4.85 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.78
CHH), 4.54 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.45 (d, J = 11.8 Hz, (d, J = 11.6 Hz, 1H, Bn-CHH), 4.70 (d, J = 11.4 Hz, 1H, Bn-
1H, Bn-CHH), 4.32 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.16 (dd, J = CHH), 4.36 (d, J = 11.2 Hz, 1H, Bn-CHH), 4.48 (d, J = 11.8 Hz,
7.2 Hz, J = 6.0 Hz, 1H, H-3′), 4.12 (dd, J = 7.2 Hz, J = 4.9 Hz, 1H, Bn-CHH), 4.43 (d, J = 11.8 Hz, 1H, Bn-CHH), 4.28 (d, J =
1H, H-2′), 3.85 (dd, J = 9.4 Hz, J = 5.9 Hz, 1H, H-4′), 3.81 (m, 11.4 Hz, 1H, Bn-CHH), 4.29 (m, 1H, H-2′), 4.10 (dd, J = 1.9 Hz,
1H, H-5′), 3.70 (dd, J = 10.8 Hz, J = 3.8 Hz, 1H, H-6′a), 3.59 (dd, J = 1.9 Hz, 1H, H-4′), 3.86 (dd, J = 6.3 Hz, J = 6.3 Hz, 1H, H-5′),
J = 10.8 Hz, J = 2.1 Hz, 1H, H-6′b). 13C NMR (100 MHz, CDCl3): 3.81 (dd, J = 9.4 Hz, J = 2.5 Hz, 1H, H-3′), 3.66–3.58 (m, 2H,
δ 152.0 (C-2), 151.6 (C-4), 150.9 (C-6), 144.9 (C-8), 138.1 H-6′a and H-6′b). 13C NMR (100 MHz, CDCl3): δ 161.6 (C-4),
(Bn-Cq), 137.7 (Bn-Cq), 137.7 (Bn-Cq), 136.3 (Bn-Cq), 131.3 152.1 (C-2), 147.2 (C-8), 143.2 (C-6), 138.2 (Bn-Cq), 137.5 (Bn-
(C-5), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), Cq), 137.4 (Bn-Cq), 136.2 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5
128.4 (Bn), 128.3 (Bn), 128.3 (Bn), 128.2 (Bn), 128.0 (Bn), 128.0 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.2 (Bn), 128.2 (Bn),
(Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9
127.9 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 79.8 (C-3′), 79.4 (Bn), 127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.6 (Bn),
(C-1′), 76.3 (C-2′), 76.1 (C-4′), 73.6 (C-5′), 74.1 (Bn-CH2), 74.0 127.6 (Bn), 122.3 (C-5), 85.2 (C-1′), 83.4 (C-3′), 77.2 (C-2′), 76.6
(Bn-CH2), 73.5 (Bn-CH2), 73.2 (Bn-CH2), 67.9 (C-6′). Elemental (C-5′), 75.0 (Bn-CH2), 74.9 (Bn-CH2), 73.6 (Bn-CH2), 73.1 (C-4),
anal. calcd for C39H37ClN4O5: C, 69.17; H, 5.51; N, 8.27; found: 72.6 (Bn-CH2), 68.2 (C-6′). Elemental anal. calcd for
C, 68.84; H, 5.70; N, 8.03.
C39H37ClN4O5: C, 69.17; H, 5.51; N, 8.27; found: C, 68.97; H,
5.69; N, 7.99.
Data for compound 8. Colourless oil; Rf = 0.43 (EtOAc–hexane
Data for compound 6. Colourless crystals; mp 146–149 °C;
Rf = 0.35 (EtOAc–hexane 1 : 2). [α]2D0 −24° (c 1.03, CHCl3).
ESI-MS m/z (%): 677.1 ([M + H]+, 100), 699.3 ([M + Na]+, 29), 1 : 2). [α]D20 −14° (c 1.00, CHCl3). ESI-MS m/z (%): 587.0 ([M −
1
1354.7 ([2M + H]+, 12). H NMR (400 MHz, CDCl3): δ 8.68 (s, Bn + H]+, 72), 609.3 ([M − Bn + Na]+, 62), 677.0 ([M + H]+, 100),
1H, H-2), 8.03 (s, 1H, H-8), 7.39–7.27 (m, 13H, BnH), 7.23–7.18 1354.8 ([2M + H]+, 29). H NMR (400 MHz, CDCl3): δ 8.65 (s,
1
(m, 2H, BnH), 7.12 (m, 1H, BnH), 6.99 (m, 2H, BnH), 5.59 (d, 1H, H-2), 8.38 (s, 1H, H-8), 7.44–7.28 (m, 14H, BnH), 7.18–7.09
J = 9.0 Hz, 1H, H-1′), 4.99 (d, J = 11.0 Hz, 1H, Bn-CHH), 4.94 (d, (m, 4H, BnH), 6.85 (m, 2H, BnH), 6.29 (d, J = 3.0 Hz, 1H, H-1′),
J = 11.0 Hz, 1H, Bn-CHH), 4.88 (d, J = 10.7 Hz, 1H, Bn-CHH), 4.82 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.71 (d, J = 11.7 Hz, 1H, Bn-
4.65 (d, J = 10.7 Hz, 1H, Bn-CHH), 4.61 (d, J = 11.7 Hz, 1H, CHH), 4.69 (d, J = 11.7 Hz, 1H, Bn-CHH), 4.64 (d, J = 12.1 Hz,
Bn-CHH), 4.55 (d, J = 12.2 Hz, 1H, Bn-CHH), 4.48 (d, J = 12.2 1H, Bn-CHH), 4.53 (d, J = 14.4 Hz, 1H, Bn-CHH), 4.51 (d, J =
Hz, 1H, Bn-CHH), 4.17 (d, J = 11.7 Hz, 1H, Bn-CHH), 4.07 (dd, 14.4 Hz, 1H, Bn-CHH), 4.41 (d, J = 11.9 Hz, 1H, Bn-CHH), 4.39
J = 9.0 Hz, J = 9.0 Hz, 1H, H-2′), 3.91 (dd, J = 9.0 Hz, J = 8.3 Hz, (m, 1H, H-5′), 4.21 (dd, J = 4.9 Hz, J = 2.9 Hz, 1H, H-4′), 4.13 (d,
1H, H-3′), 3.87 (dd, J = 9.6 Hz, J = 9.1 Hz, 1H, H-4′), 3.77–3.68 J = 11.9 Hz, 1H, Bn-CHH), 4.09–4.06 (m, 2H, H-6′a and H-3′),
(m, 3H, H-6′a and H-5′ and H-6′b). 13C NMR (100 MHz, CDCl3): 3.93 (dd, J = 5.5 Hz, J = 3.1 Hz, 1H, H-2′), 3.82 (dd, J = 11.3 Hz,
δ 152.0 (C-2), 151.4 (C-4), 151.0 (C-6), 143.2 (C-8), 137.9 (Bn- J = 3.8 Hz, 1H, H-6′b). 13C NMR (100 MHz, CDCl3): δ 151.6
Cq), 137.7 (Bn-Cq), 137.6 (Bn-Cq), 136.2 (Bn-Cq), 131.5 (C-5), (C-2), 150.8 (C-4), 150.5 (C-6), 145.2 (C-8), 137.8 (Bn-Cq), 137.8
128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn-Cq), 137.6 (Bn-Cq), 136.0 (Bn-Cq), 131.1 (C-5), 128.5 (Bn),
(Bn), 128.1 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.3 (Bn), 128.3 (Bn), 128.2
127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 128.2 (Bn), 128.2 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn),
(Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 85.9 (C-3′), 83.3 (C-1′), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7
79.9 (C-2′), 78.3 (C-5′), 77.2 (C-4′), 75.9 (Bn-CH2), 75.3 (Bn- (Bn), 127.7 (Bn), 127.6 (Bn), 77.2 (C-1′), 75.5 (C-5′), 74.4 (C-2′),
CH2), 74.9 (Bn-CH2), 73.5 (Bn-CH2), 68.2 (C-6′). Elemental anal. 74.4 (C-3′), 73.4 (Bn-CH2), 73.3 (Bn-CH2), 73.2 (Bn-CH2), 72.8
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(Bn-CH2), 72.6 (C-4′), 65.8 (C-6′). Elemental anal. calcd for 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 122.6 (C-5), 78.6
C39H37ClN4O5: C, 69.17; H, 5.51; N, 8.27; found: C, 69.13; H, (C-1′), 74.1 (C-3′), 77.1 (C-5′), 75.4 (C-2′), 73.3 (Bn-CH2), 73.3
5.59; N, 8.13.
(Bn-CH2), 71.9 (Bn-CH2), 73.5 (C-4′), 71.3 (Bn-CH2), 67.5 (C-6′).
Elemental anal. calcd for C39H37ClN4O5: C, 69.17; H, 5.51;
N, 8.27; found: C, 68.96; H, 5.63; N, 8.11.
Data for compound 9. Colourless oil; Rf = 0.30 (EtOAc–
hexane 1 : 2). [α]2D0 −16° (c 0.92, CHCl3). ESI-MS m/z (%): 497.1
([M − 2Bn + H]+, 8), 519.3 ([M − 2Bn + Na]+, 12), 587.1 ([M −
Data for compound 11. Colourless oil; Rf = 0.31 (EtOAc–
Bn + H]+, 23), 609.4 ([M − Bn + Na]+, 24), 677.1 ([M + H]+, 100), hexane 1 : 1). [α]D20 +62° (c 1.02, CHCl3). ESI-MS m/z (%): 587.1
699.3 ([M + Na]+, 49). 1H NMR (400 MHz, CDCl3): δ 8.72 (s, 1H, ([M − Bn + H]+, 100), 609.3 ([M − Bn + Na]+, 29), 677.0 ([M +
H-2), 8.07 (s, 1H, H-8), 7.44–7.24 (m, 15H, BnH), 7.16 (m, 1H, H]+, 4). H NMR (400 MHz, CDCl3): δ 8.77 (s, 1H, H-2), 8.51 (s,
1
BnH), 7.03 (m, 2H, BnH), 6.64 (m, 2H, BnH), 5.68 (d, J = 9.0 1H, H-8), 7.46–7.23 (m, 15H, BnH), 6.92–6.86 (m, 3H, BnH),
Hz, 1H, H-1′), 5.05 (d, J = 11.5 Hz, 1H, Bn-CHH), 4.84 (d, J = 6.73 (m, 2H, BnH), 6.90 (d, J = 1.0 Hz, 1H, H-1′), 4.98 (d, J =
11.7 Hz, 1H, Bn-CHH), 4.79 (d, J = 11.7 Hz, 1H, Bn-CHH), 4.70 10.8 Hz, 1H, Bn-CHH), 4.90 (d, J = 11.7 Hz, 1H, Bn-CHH), 4.80
(d, J = 11.5 Hz, 1H, Bn-CHH), 4.69 (d, J = 11.6 Hz, 1H, (d, J = 11.7 Hz, 1H, Bn-CHH), 4.68 (d, J = 11.8 Hz, 1H,
Bn-CHH), 4.48 (d, J = 11.9 Hz, 1H, Bn-CHH), 4.43 (d, J = 11.9 Bn-CHH), 4.66 (d, J = 10.8 Hz, 1H, Bn-CHH), 4.64 (d, J =
Hz, 1H, Bn-CHH), 4.32 (dd, J = 9.2 Hz, J = 9.0 Hz, 1H, H-2′), 12.4 Hz, 1H, Bn-CHH), 4.56 (d, J = 12.4 Hz, 1H, Bn-CHH), 4.07
4.24 (d, J = 14.4 Hz, 1H, Bn-CHH), 4.09 (m, 1H, H-4′), 3.86 (m, (dd, J = 9.5 Hz, J = 9.5 Hz, 1H, H-4′), 4.02 (dd, J = 2.5 Hz, J = 1.0
2H, H-5′ and H-3′), 3.61–3.59 (m, 2H, H-6′a and H-6′b). 13C Hz, 1H, H-2′), 3.84 (dd, J = 9.5 Hz, J = 2.5 Hz, 1H, H-3′), 3.78
NMR (100 MHz, CDCl3): δ 152.0 (C-2), 151.6 (C-4), 150.8 (C-6), (m, 2H, H-6′a and H-6′b), 3.71 (ddd, J = 9.5 Hz, J = 3.6 Hz, J =
143.0 (C-8), 138.3 (Bn-Cq), 137.7 (Bn-Cq), 137.4 (Bn-Cq), 136.4 3.6 Hz, 1H, H-5′), 4.34 (d, J = 11.8 Hz, 1H, Bn-CHH). 13C NMR
(Bn-Cq), 131.1 (C-5), 128.6 (Bn), 128.6 (Bn), 128.4 (Bn), 128.4 (100 MHz, CDCl3): δ 161.8 (C-4), 151.8 (C-2), 147.9 (C-8), 141.1
(Bn), 128.3 (Bn), 128.3 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), (C-6), 137.8 (Bn-Cq), 137.7 (Bn-Cq), 137.4 (Bn-Cq), 135.6 (Bn-
127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 Cq), 128.8 (Bn), 128.8 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn),
(Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.6 (Bn), 127.6 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.3 (Bn), 128.1 (Bn), 128.1
83.4 (C-3′), 82.8 (C-1′), 77.7 (C-2′), 76.6 (C-5′), 75.1 (Bn-CH2), (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn),
74.7 (Bn-CH2), 73.6 (Bn-CH2), 73.2 (C-4′), 72.8 (Bn-CH2), 68.2 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 120.4 (C-5), 83.9
(C-6′). Elemental anal. calcd for C39H37ClN4O5: C, 69.17; H, (C-1′), 82.3 (C-3′), 75.4 (Bn-CH2), 74.1 (Bn-CH2), 74.0 (Bn-CH2),
5.51; N, 8.27; found: C, 69.00; H, 5.77; N, 8.17.
6-Chloro-7-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)purine Elemental anal. calcd for C39H37ClN4O5: C, 69.17; H, 5.51;
(10), 6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)- N, 8.27; found: C, 69.04; H, 5.62; N, 8.17.
73.6 (C-5′), 73.5 (Bn-CH2), 72.8 (C-2′), 73.5 (C-4′), 67.8 (C-6′).
Data for compound 12. Colourless oil; Rf = 0.27 (EtOAc–
purine (11), 6-chloro-9-(2,3,4,6-tetra-O-benzyl-α-D-mannopyrano-
syl)purine (12) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-manno-
pyranosyl)purine (13). The compounds were obtained by
the reaction of methyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyrano-
side (250 mg, 0.45 mmol) and CP (104 mg, 0.67 mmol) accord-
ing to the general procedure. Purification by column
chromatography (EtOAc–cyclohexane 1 : 1) yielded 10
(52.2 mg; 17%), 11 (42.8 mg; 14%), 12 (82.6 mg; 27%) and 13
(30.6 mg; 10%)
hexane 1 : 2). [α]2D0 +64° (c 1.58, CHCl3). ESI-MS m/z (%): 677.0
([M + H]+, 100), 699.1 ([M + Na]+, 26), 1354.8 ([2M + 2H]+, 23),
1
1374.9 ([2M + Na]+, 18). H NMR (400 MHz, CDCl3): δ 8.60 (s,
1H, H-2), 8.11 (s, 1H, H-8), 7.39–7.20 (m, 15H, BnH), 7.16 (m,
1H, BnH), 7.09 (m, 2H, BnH), 6.88 (m, 2H, BnH), 6.03 (d, J =
8.1 Hz, 1H, H-1′), 4.76 (dd, J = 8.1 Hz, J = 2.6 Hz, 1H, H-2′),
4.72 (d, J = 12.3 Hz, 1H, Bn-CHH), 4.65 (d, J = 12.3 Hz, 1H, Bn-
CHH), 4.52 (d, J = 12.2 Hz, 1H, Bn-CHH), 4.50–4.42 (m, 5H, Bn-
Data for compound 10. Colourless oil; Rf = 0.46 (EtOAc–cyclo- CH2 and H-5′), 4.16 (d, J = 12.2 Hz, 1H, Bn-CHH), 3.99 (dd, J =
hexane 1 : 1). [α]2D0 +82° (c 0.86, CHCl3). ESI-MS m/z (%): 676.9 2.8 Hz, J = 2.8 Hz, 1H, H-3′), 3.86 (dd, J = 5.8 Hz, J = 3.2 Hz,
([M + H]+, 100), 699.3 ([M + Na]+, 6). 1H NMR (400 MHz, 1H, H-4′), 3.75 (dd, J = 10.8 Hz, J = 6.0 Hz, 1H, H-6′a), 3.65 (dd,
CDCl3): δ 8.87 (s, 1H, H-2), 8.28 (s, 1H, H-8), 7.41–7.12 (m, J = 10.8 Hz, J = 3.9 Hz, 1H, H-6′b). 13C NMR (100 MHz, CDCl3):
18H, BnH), 6.82 (m, 2H, BnH), 6.49 (d, J = 9.1 Hz, 1H, H-1′), δ 151.7 (C-2), 151.4 (C-4), 150.9 (C-6), 144.9 (C-8), 137.8 (Bn-
4.69 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.62 (d, J = 4.3 Hz, 1H, Bn- Cq), 137.6 (Bn-Cq), 137.5 (Bn-Cq), 136.4 (Bn-Cq), 132.0 (C-5),
CHH), 4.59 (d, J = 4.3 Hz, 1H, Bn-CHH), 4.50 (s, 2H, Bn-CH2), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.3
4.44 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.39 (dd, J = 6.6 Hz, J = (Bn), 128.3 (Bn), 128.2 (Bn), 128.2 (Bn), 128.1 (Bn), 128.1 (Bn),
6.6 Hz, 1H, H-5′), 4.28 (d, J = 11.9 Hz, 1H, Bn-CHH), 4.12 (dd, 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9
J = 9.1 Hz, J = 2.8 Hz, 1H, H-2′), 4.06 (dd, J = 2.8 Hz, J = 2.8 Hz, (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 80.9 (C-1′), 75.8 (C-5′),
1H, H-3′), 4.02 (d, J = 11.9 Hz, 1H, Bn-CHH), 3.94 (dd, J = 10.3 74.9 (C-4′), 74.1 (C-3′), 73.2 (Bn-CH2), 72.8 (Bn-CH2), 72.7 (Bn-
Hz, J = 7.6 Hz, 1H, H-6′a), 3.83 (dd, J = 3.5 Hz, J = 1.3 Hz, 1H, CH2), 72.1 (C-2′), 71.6 (Bn-CH2), 68.3 (C-6′). Elemental anal.
H-4′), 3.71 (dd, J = 10.3 Hz, J = 5.9 Hz, 1H, H-6′b). 13C NMR calcd for C39H37ClN4O5: C, 69.17; H, 5.51; N, 8.27; found: C,
(100 MHz, CDCl3): δ 161.6 (C-4), 152.2 (C-2), 146.9 (C-8), 143.4 69.00; H, 5.64; N, 7.97.
Data for compound 13. Colourless oil; Rf = 0.24 (EtOAc–
hexane 1 : 2). [α]2D0 +56° (c 0.91, CHCl3). ESI-MS m/z (%): 677.1
([M + H]+, 100), 699.3 ([M + Na]+, 38). 1H NMR (400 MHz,
CDCl3): δ 8.55 (s, 1H, H-2), 8.38 (s, 1H, H-8), 7.43–7.20 (m,
(C-6), 137.6 (Bn-Cq), 137.4 (Bn-Cq), 137.3 (Bn-Cq), 136.0 (Bn-
Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn),
128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.3 (Bn), 128.2 (Bn), 128.1
(Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn),
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15H, BnH), 7.06–6.98 (m, 3H, BnH), 6.86 (m, 2H, BnH), 5.81 7.39–7.17 (m, 15H, BnH), 7.10–6.99 (m, 3H, BnH), 6.80 (m, 2H,
(d, J = 1.1 Hz, 1H, H-1′), 4.95 (d, J = 10.8 Hz, 1H, Bn-CHH), 4.84 BnH), 5.61 (br, 1H, H-1′), 5.00 (d, J = 10.9 Hz, 1H, Bn-CHH),
(m, 2H, Bn-CH2), 4.76 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.65 (d, J = 4.94 (d, J = 10.9 Hz, 1H, Bn-CHH), 4.88 (d, J = 10.7 Hz, 1H, Bn-
10.8 Hz, 1H, Bn-CHH), 4.62 (d, J = 12.3 Hz, 1H, Bn-CHH), 4.56 CHH), 4.64 (d, J = 11.5 Hz, 1H, Bn-CHH), 4.63 (d, J = 10.7 Hz,
(d, J = 12.3 Hz, 1H, Bn-CHH), 4.33 (d, J = 11.6 Hz, 1H, 1H, Bn-CHH), 4.54 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.48 (d, J =
Bn-CHH), 4.09 (m, 2H, H-2′ and H-4′), 3.91 (dd, J = 9.4 Hz, J = 12.1 Hz, 1H, Bn-CHH), 4.24 (d, J = 11.5 Hz, 1H, Bn-CHH),
2.6 Hz, 1H, H-3′), 3.77–3.72 (m, 3H, H-6′a and H-6′b and H-5′). 3.94–3.84 (m, 3H, H-2′ and H-3′ and H-5′), 3.77–3.67 (m, 3H,
13C NMR (100 MHz, CDCl3): δ 151.3 (C-2), 150.6 (C-6), 149.8 H-4′ and H-6′a and H-6′b), 2.64 (s, 3H, Ac-Me). 13C NMR
(C-4), 144.6 (C-8), 137.8 (Bn-Cq), 137.8 (Bn-Cq), 137.6 (Bn-Cq), (100 MHz, CDCl3): δ 167.6 (Ac-COO), 162.8 (C-4), 152.1 (C-2),
136.0 (Bn-Cq), 130.7 (C-5), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 149.3 (C-8), 143.6 (C-6), 137.7 (Bn-Cq), 137.5 (Bn-Cq), 136.6
128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn-Cq), 136.0 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn),
(Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.3 (Bn), 128.3 (Bn), 128.1
127.9 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 127.6 (Bn), 127.6 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn),
(Bn), 83.1 (C-3′), 82.1 (C-1′), 78.8 (C-5′), 75.5 (Bn-CH2), 74.1 127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7
(C-4′), 74.4 (Bn-CH2), 73.5 (Bn-CH2), 73.2 (Bn-CH2), 72.6 (C-2′), (Bn), 117.9 (C-5), 85.9 (C-1′), 85.9 (C-3′), 78.1 (C-4′), 77.2 (C-2′),
68.8 (C-6′). Elemental anal. calcd for C39H37ClN4O5: C, 69.17; 77.2 (C-5′), 75.9 (Bn-CH2), 75.3 (Bn-CH2), 74.7 (Bn-CH2), 73.5
H, 5.51; N, 8.27; found: C, 69.04; H, 5.76; N, 8.04.
(Bn-CH2), 68.3 (C-6′), 25.2 (Ac-Me). Elemental anal. calcd for
2-(Acetamido)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-α-D-glucopyra- C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C, 66.81; H,
nosyl)purine (14), 2-(acetamido)-6-chloro-7-(2,3,4,6-tetra-O- 5.52; N, 9.33.
Data for compound 16. Colourless oil; Rf = 0.53 (EtOAc–
hexane 1 : 1). [α]2D0 −24° (c 1.01, CHCl3). ESI-MS m/z (%): 734.2
([M + H]+, 88), 756.3 ([M + Na]+, 100), 1468.9 ([2M + H]+, 28),
benzyl-β-D-glucopyranosyl)purine (15) and 2-(acetamido)-6-
chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (16).
The compounds were obtained by the reaction of methyl
2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (39 mg, 0.07 mmol)
and ACP (23 mg, 0.11 mmol) according to the general pro-
cedure. Purification by column chromatography (EtOAc–cyclo-
hexane 1 : 1) yielded 14 (3.6 mg; 7%), 15 (17.5 mg; 34%) and 16
(14.9 mg; 29%).
1
1489.7 ([2M + Na]+, 10). H NMR (400 MHz, CDCl3): δ 8.05 (s,
1H, NH), 8.92 (s, 1H, H-8), 7.39–7.18 (m, 15H, BnH), 7.12–6.98
(m, 3H, BnH), 6.71 (m, 2H, BnH), 5.38 (d, J = 9.1 Hz, 1H, H-1′),
5.00 (d, J = 11.0 Hz, 1H, Bn-CHH), 4.94 (d, J = 11.0 Hz, 1H, Bn-
CHH), 4.88 (d, J = 10.7 Hz, 1H, Bn-CHH), 4.64 (d, J = 10.7 Hz,
Data for compound 14. Yellow crystals; mp 81–84 °C; Rf = 1H, Bn-CHH), 4.61 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.52 (d, J =
0.23 (EtOAc–hexane 1 : 1). [α]2D0 +33° (c 0.88, CHCl3). ESI-MS 12.1 Hz, 1H, Bn-CHH), 4.45 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.24
m/z (%): 734.2 ([M + H]+, 100), 756.3 ([M + Na]+, 54), 1467.1 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.11 (dd, J = 9.0 Hz, J = 9.0 Hz,
([2M + H]+, 34). 1H NMR (400 MHz, CDCl3): δ 8.61 (s, 1H, H-8), 1H, H-2′), 3.87 (dd, J = 9.0 Hz, J = 9.0 Hz, 1H, H-3′), 3.80 (dd,
8.06 (s, 1H, NH), 7.42–7.24 (m, 12H, BnH), 7.21–7.08 (m, 6H, J = 9.0 Hz, J = 9.0 Hz, 1H, H-4′), 3.75–3.68 (m, 3H, H-6′a and
BnH), 6.96 (m, 2H, BnH), 6.24 (d, J = 4.8 Hz, 1H, H-1′), 4.86 (d, H-6′b and H-5′), 2.50 (s, 3H, Ac-Me). 13C NMR (100 MHz,
J = 11.2 Hz, 1H, Bn-CHH), 4.81 (d, J = 11.2 Hz, 1H, Bn-CHH), CDCl3): δ 171.1 (Ac-COO), 151.9 (C-2), 151.8 (C-4), 151.2 (C-6),
4.75 (d, J = 11.1 Hz, 1H, Bn-CHH), 4.71 (d, J = 12.1 Hz, 1H, Bn- 143.0 (C-8), 137.9 (Bn-Cq), 137.5 (Bn-Cq), 137.5 (Bn-Cq),
CHH), 4.56 (d, J = 11.1 Hz, 1H, Bn-CHH), 4.55 (d, J = 12.2 Hz, 136.3 (Bn-Cq), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn),
1H, Bn-CHH), 4.48 (d, J = 12.2 Hz, 1H, Bn-CHH), 4.36 (d, J = 128.4 (Bn), 128.4 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0
12.1 Hz, 1H, Bn-CHH), 4.09 (dd, J = 6.7 Hz, J = 4.8 Hz, 1H, (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn),
H-2′), 4.04 (dd, J = 7.4 Hz, J = 6.7 Hz, 1H, H-3′), 3.84 (dd, J = 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (C-5), 127.7 (Bn), 127.7
9.5 Hz, J = 6.7 Hz, 1H, H-4′), 3.66 (dd, J = 10.6 Hz, J = 3.5 Hz, (Bn), 85.9 (C-3′), 83.8 (C-1′), 75.1 (C-2′), 77.9 (C-5′), 77.2 (C-4′),
1H, H-6′a), 3.56 (m, 2H, H-5′ and H-6′b), 2.63 (s, 3H, Ac-Me). 75.8 (Bn-CH2), 75.2 (Bn-CH2), 74.5 (Bn-CH2), 73.5 (Bn-CH2),
13C NMR (100 MHz, CDCl3): δ 167.1 (Ac-COO), 162.8 (C-4), 68.2 (C-6′), 25.2 (Ac-Me). Elemental anal. calcd for
152.4 (C-2), 149.3 (C-8), 143.6 (C-6), 137.5 (Bn-Cq), 137.5 (Bn- C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C, 66.81; H,
Cq), 137.5 (Bn-Cq), 136.2 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5 5.70; N, 9.42.
(Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn),
2-(Acetamido)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-α-D-galacto-
128.4 (Bn), 128.2 (Bn), 128.2 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 pyranosyl)purine (17), 2-(acetamido)-6-chloro-7-(2,3,4,6-tetra-
(Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.8 (Bn), 127.7 (Bn), O-benzyl-β-D-galactopyranosyl)purine (18), 2-(acetamido)-6-
127.7 (Bn), 118.2 (C-5), 81.0 (C-1′), 80.8 (C-3′), 76.9 (C-2′), 76.6 chloro-9-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)purine (19)
(C-4′), 74.6 (Bn-CH2), 74.3 (Bn-CH2), 74.0 (Bn-CH2), 73.5 (Bn- and 2-(acetamido)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galacto-
CH2), 73.1 (C-5′), 68.0 (C-6′), 25.2 (Ac-Me). Elemental anal. pyranosyl)purine (20). The compounds were obtained by
calcd for C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C, the reaction of methyl 2,3,4,6-tetra-O-benzyl-α-D-galactopyrano-
66.87; H, 5.63; N, 9.32.
side (600 mg, 1.08 mmol) and ACP (340 mg, 1.62 mmol)
according to the general procedure. Purification by column
chromatography (EtOAc–cyclohexane, 1 : 1) yielded 17
(25.2 mg; 3%), 18 (346.5 mg; 44%), 19 (78.8 mg; 10%) and 20
(322.9 mg; 41%).
Data for compound 15. Yellow crystals; mp 71–74 °C; Rf =
0.09 (EtOAc–hexane 1 : 1). [α]2D0 −13° (c 0.99, CHCl3). ESI-MS
m/z (%): 734.13 ([M + H]+, 100), 756.2 ([M + Na]+, 78). H NMR
1
(400 MHz, CDCl3): δ 8.15 (s, 1H, H-8), 8.06 (s, 1H, NH),
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Data for compound 17. Yellow oil; Rf = 0.11 (EtOAc–heptane CH2), 4.52 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.44–4.28 (m, 2H, H-5′
3 : 2). [α]2D0 +3° (c 1.00, CHCl3). ESI-MS m/z (%): 734.1 ([M + H]+, and H-4′), 4.20 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.15 (m, 1H,
100), 756.3 ([M + Na]+, 28), 1467.8 ([2M + H]+, 58), 1491.1 ([2M H-3′), 4.03 (dd, J = 11.2 Hz, J = 8.3 Hz, 1H, H-6′a), 3.97 (m, 1H,
+ Na]+, 28). 1H NMR (400 MHz, CDCl3): δ 8.56 (s, 1H, H-8), 8.28 H-2′), 3.81 (dd, J = 11.2 Hz, J = 3.4 Hz, 1H, H-6′b), 2.44 (s, 3H,
(s, 1H, NH), 7.47–7.22 (m, 15H, BnH), 7.19–7.08 (m, 3H, BnH), Ac-Me). 13C NMR (100 MHz, CDCl3): δ 171.5 (Ac-COO), 152.2
6.86 (m, 2H, BnH), 6.36 (d, J = 2.4 Hz, 1H, H-1′), 4.84 (d, J = (C-2), 151.5 (C-4), 150.9 (C-6), 144.3 (C-8), 137.8 (Bn-Cq), 137.7
11.8 Hz, 1H, Bn-CHH), 4.75 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.70 (Bn-Cq), 137.7 (Bn-Cq), 136.2 (Bn-Cq), 128.5 (Bn), 128.5 (Bn),
(d, J = 11.8 Hz, 1H, Bn-CHH), 4.67 (d, J = 11.6 Hz, 1H, Bn- 128.5 (Bn), 128.5 (Bn), 128.3 (Bn), 128.3 (Bn), 128.2 (Bn), 128.2
CHH), 4.55 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.50 (d, J = 11.6 Hz, (Bn), 128.2 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn),
1H, Bn-CHH), 4.48 (d, J = 11.9 Hz, 1H, Bn-CHH), 4.28–4.20 (m, 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 127.8
2H, H-5′ and H-4′), 4.14 (d, J = 11.9 Hz, 1H, Bn-CHH), (C-5), 127.7 (Bn), 127.6 (Bn), 77.1 (C-1′), 75.4 (C-5′), 74.7 (C-3′),
4.01–3.95 (m, 3H, H-3′ and H-6′a and H-2′), 3.81 (dd, J = 11.4 74.1 (C-2′), 73.3 (Bn-CH2), 73.2 (Bn-CH2), 73.1 (Bn-CH2), 73.0
Hz, J = 3.5 Hz, 1H, H-6′b), 2.67 (s, 3H, Ac-Me). 13C NMR (Bn-CH2), 72.9 (C-4′), 65.9 (C-6′), 25.1 (Ac-Me). Elemental anal.
(100 MHz, CDCl3): δ 171.6 (Ac-COO), 162.8 (C-4), 152.1 (C-2), calcd for C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C,
149.2 (C-8), 142.6 (C-6), 137.7 (Bn-Cq), 137.6 (Bn-Cq), 137.5 69.80; H, 5.69; N, 9.41.
Data for compound 20. A colourless oil; Rf = 0.57 (EtOAc–
hexane 1 : 1). [α]2D0 −14° (c 1.02, CHCl3). ESI-MS m/z (%): 734.3
([M + H]+, 100), 756.5 ([M + Na]+, 56), 1467.9 ([2M + H]+, 22).
1H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H, NH), 7.92 (s, 1H,
H-8), 7.43–7.22 (m, 15H, BnH), 7.10 (m, 1H, BnH), 7.01 (m,
2H, BnH), 6.73 (m, 2H, BnH), 5.35 (d, J = 9.0 Hz, 1H, H-1′),
4.96 (d, J = 11.4 Hz, 1H, Bn-CHH), 4.81 (d, J = 11.7 Hz, 1H, Bn-
CHH), 4.75 (d, J = 11.7 Hz, 1H, Bn-CHH), 4.68 (d, J = 11.8 Hz,
1H, Bn-CHH), 4.64 (d, J = 11.8 Hz, 1H, Bn-CHH), 4.45 (d, J =
(Bn-Cq), 135.9 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn),
128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.3 (Bn), 128.3 (Bn), 128.3
(Bn), 128.3 (Bn), 128.2 (Bn), 128.1 (Bn), 128.0 (Bn), 127.9 (Bn),
127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7
(Bn), 117.7 (C-5), 78.5 (C-1′), 75.1 (C-5′), 74.7 (C-3′), 74.4 (C-2′),
73.4 (Bn-CH2), 73.3 (Bn-CH2), 73.1 (Bn-CH2), 73.0 (Bn-CH2),
72.5 (C-4′), 65.5 (C-6′), 25.2 (Ac-Me). Elemental anal. calcd for
C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C, 66.77; H,
5.53; N, 9.39.
Data for compound 18. Yellow oil; Rf = 0.26 (EtOAc–cyclo- 11.9 Hz, 1H, Bn-CHH), 4.43 (m, 1H, H-2′), 4.40 (d, J = 11.9 Hz,
hexane 1 : 1). [α]2D0 −4° (c 1.26, CHCl3). ESI-MS m/z (%): 734.2 1H, Bn-CHH), 4.29 (d, J = 11.4 Hz, 1H, Bn-CHH), 4.06 (m, 1H,
([M + H]+, 82), 756.4 ([M + Na]+, 22), 1468.9 ([2M + H]+, 100), H-4′), 3.79 (dd, J = 6.3 Hz, J = 6.3 Hz, 1H, H-5′), 3.76 (dd, J = 9.6
1
1491.2 ([2M + Na]+, 76). H NMR (400 MHz, CDCl3): δ 8.16 (s, Hz, J = 2.6 Hz, 1H, H-3′), 3.55 (m, 2H, H-6′a and H-6′b), 2.42 (s,
1H, H-8), 8.00 (s, 1H, NH), 7.43–7.23 (m, 15H, BnH), 7.12–7.01 3H, Ac-Me). 13C NMR (100 MHz, CDCl3): δ 171.3 (Ac-COO),
(m, 3H, BnH), 6.81 (m, 2H, BnH), 5.65 (d, J = 7.3 Hz, 1H, H-1′), 152.1 (C-2), 151.9 (C-4), 151.1 (C-6), 143.1 (C-8), 138.2 (Bn-Cq),
5.00 (d, J = 11.2 Hz, 1H, Bn-CHH), 4.84 (d, J = 11.6 Hz, 1H, 137.6 (Bn-Cq), 137.4 (Bn-Cq), 136.6 (Bn-Cq), 128.6 (Bn), 128.6
Bn-CHH), 4.77 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.71 (d, J = (Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.1 (Bn),
11.4 Hz, 1H, Bn-CHH), 4.62 (d, J = 11.2 Hz, 1H, Bn-CHH), 4.48 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0
(d, J = 11.8 Hz, 1H, Bn-CHH), 4.43 (d, J = 11.8 Hz, 1H, Bn- (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn),
CHH), 4.29 (d, J = 11.4 Hz, 1H, Bn-CHH), 4.28 (m, 1H, H-2′), 127.8 (C-5), 127.6 (Bn), 127.6 (Bn), 83.9 (C-1′), 83.6 (C-3′), 76.5
4.09 (d, J = 2.5 Hz, 1H, H-4′), 3.84 (m, 1H, H-5′), 3.79 (dd, J = (C-5′), 75.7 (C-2′), 74.8 (Bn-CH2), 74.8 (Bn-CH2), 73.6 (Bn-CH2),
9.3 Hz, J = 2.5 Hz, 1H, H-3′), 3.66–3.57 (m, 2H, H-6′a and H-6′ 73.0 (C-4′), 72.6 (Bn-CH2), 68.2 (C-6′), 25.2 (Ac-Me). Elemental
b), 2.63 (s, 3H, Ac-Me). 13C NMR (100 MHz, CDCl3): δ 171.4 anal. calcd for C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found:
(Ac-COO), 162.9 (C-4), 152.0 (C-2), 147.9 (C-8), 143.7 (C-6), C, 66.92; H, 5.61; N, 9.47.
138.1 (Bn-Cq), 137.5 (Bn-Cq), 137.3 (Bn-Cq), 136.0 (Bn-Cq),
2-(Acetamido)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-α-D-manno-
128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 pyranosyl)purine (21), 2-(acetamido)-6-chloro-7-(2,3,4,6-tetra-
(Bn), 128.2 (Bn), 128.2 (Bn), 128.1 (Bn), 128.1 (Bn), 128.0 (Bn), O-benzyl-β-D-mannopyranosyl)purine (22), 2-(acetamido)-6-
128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 chloro-9-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl)purine (23)
(Bn), 127.9 (Bn), 127.6 (Bn), 127.6 (Bn), 118.7 (C-5), 85.2 (C-1′), and 2-(acetamido)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-manno-
83.6 (C-3′), 77.2 (C-2′), 76.5 (C-5′), 75.1 (Bn-CH2), 75.0 (Bn- pyranosyl)purine (24). The compounds were obtained by the
CH2), 73.6 (Bn-CH2), 73.2 (C-4′), 72.6 (Bn-CH2), 68.2 (C-6′), 25.2 reaction of methyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside
(Ac-Me). Elemental anal. calcd for C41H40ClN5O6: C, 67.07; H, (250 mg, 0.45 mmol) and ACP (112 mg, 0.54 mmol) according
5.49; N, 9.54; found: C, 67.00; H, 5.67; N, 9.40.
to the general procedure. Purification by column chromato-
graphy (EtOAc–cyclohexane 1 : 1) yielded 21 (27.1 mg; 8%), 22
(92.5 mg; 28%), 23 (81.3 mg; 24%) and 24 (10.2 mg, 3%).
Data for compound 21. Yellow oil; Rf = 0.47 (EtOAc–hexane
Data for compound 19. Yellow oil; Rf = 0.56 (EtOAc–heptane
3 : 2). [α]2D0 +5° (c 0.99, CHCl3). ESI-MS m/z (%): 734.1 ([M + H]+,
100), 756.4 ([M + Na]+, 54), 1467.5 ([2M + H]+, 38), 1492.1 ([2M
+ Na]+, 22). 1H NMR (400 MHz, CDCl3): δ 8.25 (s, 1H, H-8), 8.04 4 : 1). [α]D20 +38° (c 1.03, CHCl3). ESI-MS m/z (%): 734.1 ([M +
(s, 1H, NH), 7.47–7.12 (m, 18H, BnH), 6.89 (m, 2H, BnH), 6.08 H]+, 100), 756.3 ([M + Na]+, 58), 1467.9 ([2M + H]+, 20), 1488.7
(d, J = 3.0 Hz, 1H, H-1′), 4.86 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.77 ([2M + Na]+, 8). H NMR (400 MHz, CDCl3): δ 8.16 (s, 1H, H-8),
1
(d, J = 11.8 Hz, 1H, Bn-CHH), 4.71 (d, J = 12.0 Hz, 1H, Bn- 8.02 (s, 1H, NH), 7.41–7.14 (m, 18H, BnH), 6.88 (m, 2H, BnH),
CHH), 4.68 (d, J = 11.8 Hz, 1H, Bn-CHH), 4.53 (m, 2H, Bn- 6.36 (d, J = 8.9 Hz, 1H, H-1′), 4.68 (d, J = 12.0 Hz, 1H, Bn-CHH),
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4.60 (d, J = 12.0 Hz, 1H, Bn-CHH), 4.59 (d, J = 11.6 Hz, 1H, Bn- 152.1 (C-2), 151.5 (C-4), 151.2 (C-6), 144.5 (C-8), 137.8 (Bn-Cq),
CHH), 4.50 (s, 2H, Bn-CH2), 4.44 (d, J = 11.6 Hz, 1H, Bn-CHH), 137.5 (Bn-Cq), 137.3 (Bn-Cq), 136.5 (Bn-Cq), 128.6 (Bn), 128.6
4.37 (m, 1H, H-5′), 4.30 (d, J = 11.9 Hz, 1H, Bn-CHH), 4.11 (dd, (Bn), 128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn),
J = 8.9 Hz, J = 2.5 Hz, 1H, H-2′), 4.05 (m, 1H, H-3′), 4.04 (d, J = 128.3 (Bn), 128.2 (Bn), 128.2 (Bn), 128.2 (C-5), 128.1 (Bn), 128.1
11.9 Hz, 1H, Bn-CHH), 3.92 (dd, J = 10.2 Hz, J = 7.5 Hz, 1H, (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn),
H-6′a), 3.82 (dd, J = 3.3 Hz, J = 1.2 Hz, 1H, H-4′), 3.69 (dd, J = 127.7 (Bn), 127.6 (Bn), 127.6 (Bn), 80.7 (C-1′), 76.0 (C-5′), 74.7
10.2 Hz, J = 5.9 Hz, 1H, H-6′b), 2.63 (s, 3H, Ac-Me). 13C NMR (C-4′), 73.6 (C-3′), 73.1 (C-2′), 72.7 (Bn-CH2), 72.1 (Bn-CH2),
(100 MHz, CDCl3): δ 171.5 (Ac-COO), 162.9 (C-4), 152.1 (C-2), 71.9 (Bn-CH2), 71.2 (Bn-CH2), 68.1 (C-6′), 25.1 (Ac-Me). Elemen-
147.6 (C-8), 143.8 (C-6), 137.6 (Bn-Cq), 137.4 (Bn-Cq), 137.3 tal anal. calcd for C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54;
(Bn-Cq), 136.1 (Bn-Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), found: C, 66.91; H, 5.52; N, 9.41.
Data for compound 24. Yellow oil; Rf = 0.61 (EtOAc–hexane
1 : 1). [α]2D0 +77° (c 1.00, CHCl3). ESI-MS m/z (%): 734.5 ([M +
H]+, 100), 756.6 ([M + Na]+, 52). 1H NMR (400 MHz, CDCl3):
δ 8.21 (s, 1H, H-8), 8.19 (s, 1H, NH), 7.38–7.16 (m, 15H, BnH),
7.03–6.96 (m, 3H, BnH), 6.83 (m, 2H, BnH), 5.62 (br, 1H, H-1′),
4.89 (d, J = 10.8 Hz, 1H, Bn-CHH), 4.79 (m, 2H, Bn-CH2), 4.72
(d, J = 11.6 Hz, 1H, Bn-CHH), 4.59 (d, J = 10.8 Hz, 1H, Bn-
CHH), 4.56 (d, J = 12.2 Hz, 1H, Bn-CHH), 4.49 (d, J = 12.2 Hz,
128.5 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn), 128.4 (Bn), 128.2
(Bn), 128.1 (Bn), 128.1 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn),
127.9 (Bn), 127.8 (Bn), 127.8 (Bn), 127.8 (Bn), 127.7 (Bn), 127.7
(Bn), 119.1 (C-5), 78.6 (C-1′), 77.2 (C-5′), 75.1 (C-2′), 74.1 (C-4′),
73.5 (C-3′), 73.3 (Bn-CH2), 73.3 (Bn-CH2), 71.9 (Bn-CH2), 71.3
(Bn-CH2), 67.4 (C-6′), 25.2 (Ac-Me). Elemental anal. calcd for
C41H40ClN5O6: C, 67.07; H, 5.49; N, 9.54; found: C, 66.82; H,
5.54; N, 9.31.
Data for compound 22. Yellow crystals; mp 80–82 °C; Rf = 1H, Bn-CHH), 4.32 (d, J = 11.6 Hz, 1H, Bn-CHH), 4.05 (m, 1H,
0.36 (EtOAc–hexane 4 : 1). [α]2D0 +74° (c 0.98, CHCl3). ESI-MS H-2′), 4.02 (dd, J = 9.5 Hz, J = 9.5 Hz, 1H, H-4′), 3.84 (dd, J = 9.4
m/z (%): 734.5 ([M + H]+, 70), 756.5 ([M + Na]+, 20), 1469.2 ([2M Hz, J = 2.5 Hz, 1H, H-3′), 3.72–3.64 (m, 3H, H-6′a and H-6′b
+ H]+, 100), 1491.3 ([2M + Na]+, 44). 1H NMR (400 MHz, and H-5′), 2.38 (s, 3H, Ac-Me). 13C NMR (100 MHz, CDCl3):
CDCl3): δ 8.34 (s, 1H, H-8), 8.09 (s, 1H, NH), 7.38–7.16 (m, δ 170.4 (Ac-COO), 151.3 (C-4), 150.8 (C-2), 150.6 (C-6), 143.9
15H, BnH), 6.89 (m, 3H, BnH), 6.71 (m, 2H, BnH), 5.75 (d, J = (C-8), 137.8 (Bn-Cq), 137.8 (Bn-Cq), 137.6 (Bn-Cq), 136.2 (Bn-
1.0 Hz, 1H, H-1′), 4.90 (d, J = 10.9 Hz, 1H, Bn-CHH), 4.83 (d, J = Cq), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5 (Bn), 128.4 (Bn),
11.8 Hz, 1H, Bn-CHH), 4.72 (d, J = 11.8 Hz, 1H, Bn-CHH), 4.61 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.3 (C-5), 128.0 (Bn), 128.0
(d, J = 11.8 Hz, 1H, Bn-CHH), 4.59 (d, J = 10.9 Hz, 1H, Bn- (Bn), 128.0 (Bn), 127.9 (Bn), 127.9 (Bn), 127.9 (Bn), 127.8 (Bn),
CHH), 4.55 (d, J = 12.4 Hz, 1H, Bn-CHH), 4.49 (d, J = 12.4 Hz, 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 127.7 (Bn), 82.8
1H, Bn-CHH), 4.26 (d, J = 11.8 Hz, 1H, Bn-CHH), 3.98 (dd, J = (C-3′), 82.1 (C-1′), 78.7 (C-5′), 75.3 (Bn-CH2), 74.3 (Bn-CH2),
9.4 Hz, J = 9.4 Hz, 1H, H-4′), 3.91 (m, 1H, H-2′), 3.75 (dd, J = 74.1 (C-4′), 73.4 (Bn-CH2), 73.1 (Bn-CH2), 72.5 (C-2′), 68.7
9.4 Hz, J = 2.5 Hz, 1H, H-3′), 3.71 (m, 3H, H-6′a and H-6′b and (C-6′), 25.0 (Ac-Me). Elemental anal. calcd for C41H40ClN5O6: C,
H-5′), 2.56 (s, 3H, Ac-Me). 13C NMR (100 MHz, CDCl3): δ 172.2 67.07; H, 5.49; N, 9.54; found: C, 66.98; H, 5.54; N, 9.37.
(Ac-COO), 162.9 (C-4), 152.9 (C-2), 148.5 (C-8), 141.5 (C-6),
Enzymatic studies
137.7 (Bn-Cq), 137.7 (Bn-Cq), 137.4 (Bn-Cq), 135.8 (Bn-Cq),
128.8 (Bn), 128.8 (Bn), 128.6 (Bn), 128.6 (Bn), 128.5 (Bn), 128.5
(Bn), 128.4 (Bn), 128.4 (Bn), 128.4 (Bn), 128.2 (Bn), 128.2 (Bn),
128.1 (Bn), 128.1 (Bn), 128.0 (Bn), 128.0 (Bn), 128.0 (Bn), 127.8
(Bn), 127.8 (Bn), 127.8 (Bn), 127.6 (Bn), 116.9 (C-5), 83.9 (C-1′),
82.3 (C-3′), 78.9 (C-5′), 74.0 (C-4′), 75.2 (Bn-CH2), 74.2
(Bn-CH2), 73.7 (Bn-CH2), 73.4 (Bn-CH2), 72.9 (C-2′), 68.7 (C-6′),
25.2 (Ac-Me). Elemental anal. calcd for C41H40ClN5O6: C,
67.07; H, 5.49; N, 9.54; found: C, 66.87; H, 5.39; N, 9.47.
Spectrophotometer and chemicals. A TECAN SpectraFluor-
Plus working on the kinetic mode and measuring the absor-
bance at 415 nm was used for the enzymatic studies.
Acetylcholinesterase (from Electrophorus electricus), 5,5′-dithio-
bis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide
were purchased from Fluka. Butyrylcholinesterase (from
equine serum) was purchased from Sigma and butyrylthiocho-
line iodide was bought from Aldrich.
Data for compound 23. Yellow oil; Rf = 0.73 (EtOAc–hexane
1 : 1). [α]2D0 +50° (c 1.22, CHCl3). ESI-MS m/z (%): 734.5 ([M +
H]+, 100), 756.5 ([M + Na]+, 47), 1467.1 ([2M + H]+, 24), 1491.1
([2M + Na]+, 34). 1H NMR (400 MHz, CDCl3): δ 7.94 (s, 1H,
H-8), 7.94 (s, 1H, NH), 7.37–7.00 (m, 18H, BnH), 6.82 (m, 2H,
BnH), 5.85 (d, J = 8.6 Hz, 1H, H-1′), 4.68 (d, J = 12.1 Hz, 1H,
Bn-CHH), 4.65 (m, 1H, H-2′), 4.62 (d, J = 12.1 Hz, 1H, Bn-
CHH), 4.48 (d, J = 12.1 Hz, 1H, Bn-CHH), 4.43 (d, J = 12.6 Hz,
1H, Bn-CHH), 4.42 (br, 2H, Bn-CH2), 4.40 (m, 1H, H-5′), 4.40
(d, J = 12.6 Hz, 1H, Bn-CHH), 4.10 (d, J = 12.1 Hz, 1H, Bn-
CHH), 3.96 (dd, J = 2.7 Hz, J = 2.7 Hz, 1H, H-3′), 3.70 (dd, J =
10.7 Hz, J = 6.3 Hz, 1H, H-6′a), 3.80 (dd, J = 5.2 Hz, J = 2.9 Hz,
1H, H-4′), 3.59 (dd, J = 10.6 Hz, J = 4.2 Hz, 1H, H-6′b), 2.34 (s,
3H, Ac-Me). 13C NMR (100 MHz, CDCl3): δ 170.5 (Ac-COO),
Solutions preparation. Preparation of 50 mM Tris-HCl
buffer pH 8.0: tris(hydroxymethyl)aminomethane (606 mg) was
dissolved in bidistilled water (100 mL) and adjusted with HCl
to a pH of 8.0 0.1. Buffer was freshly prepared and stored in
the refrigerator. AChE solution 2.005 U mL−1: the enzyme
(271 U mg−1, 0.037 mg) was dissolved in freshly prepared
buffer pH 8.0 (5 mL) containing NaN3 (0.98 mg). BChE solu-
tion 2.040 U mL−1: the enzyme (7.54 U mg−1, 1.353 mg) was
dissolved in freshly prepared buffer pH 8.0 (5 mL) containing
NaN3 (0.98 mg). DTNB solution 3 mM: DTNB (23.8 mg) was
dissolved in freshly prepared buffer pH 8.0 (20 mL) containing
NaCl (116.8 mg) and MgCl2 (38.0 mg). ATChI solution 15 mM:
ATChI (43.4 mg) was dissolved in bidistilled water (10 mL).
BTChI solution 15 mM: BTChI (47.6 mg) was dissolved in
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bidistilled water (10 mL). All solutions were stored in eppen-
dorf caps in the refrigerator or freezer, if necessary. The pure
compounds were initially dissolved in DMSO, galantamine
hydrobromide as standard was dissolved in bidistilled water.
The final concentrations for the enzymatic assay were yielded
by diluting the stock solution with bidistilled water. No inhi-
bition was detected by residual DMSO (<0.5%).
References
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