Fructose-fused γ-butyrolactones and lactams, synthesis and biological evaluation as GABA receptor ligands

Article abstract of DOI:10.1016/j.carres.2008.03.013

We describe the synthesis of sugar-fused β-disubstituted γ-butyrolactones, γ-butyrolactams and a lipophilic β-disubstituted GABA analogue as potential GABA receptor ligands, where the pharmacophore is engineered into the carbohydrate scaffold in the form

Full text of DOI:10.1016/j.carres.2008.03.013

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1840–1848
Note
Fructose-fused c-butyrolactones and lactams, synthesis
and biological evaluation as GABA receptor ligands
Ana C. Arau´jo,a Francesco Nicotra,b Barbara Costa,b Gabriella Giagnonib
and Laura Cipollab,*
aDepartamento de Quı´mica e Bioquı´mica, Universidade de Lisboa e CQB, C8, Campo Grande, 1749-016 Lisboa, Portugal
bDepartment of Biotechnology and Biosciences, University of Milano-Bicocca, P.za della Scienza 2, 20126 Milano, Italy
Received 5 February 2008; received in revised form 7 March 2008; accepted 9 March 2008
Available online 14 March 2008
Abstract—We describe the synthesis of sugar-fused b-disubstituted c-butyrolactones, c-butyrolactams and a lipophilic b-disubsti-
tuted GABA analogue as potential GABA receptor ligands, where the pharmacophore is engineered into the carbohydrate scaffold
in the form of a C-fructoside. The products were characterized for receptor binding studies of GABAA receptors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: C-Glycosides; Fructose; c-Butyrolactones; c-Butyrolactams; GABA receptors
Gamma-amino butyric acid (GABA, 1, Fig. 1) is the
primary inhibitory neurotransmitter in the mammalian
central nervous system (CNS).1 GABA operates
through multiple receptors subdivided into the ionotro-
pic GABAA and GABAC receptors and the metabotro-
pic GABAB receptor.2 These receptors are the target for
many endogenous and exogenous modulators that regu-
late normal and pathological brain mechanisms, such as
sleep, memory, epilepsy and emotions,3 and for a num-
ber of drugs4 including benzodiazepines, barbiturates
and neurosteroids. Consequently, compounds that act
at GABA receptors have considerable therapeutic inter-
est for use in a variety of neurological disorders, such as
epilepsy,5 anxiety,5b,6 schizophrenia,5b,7 stiff-person
syndrome5b,8 and Huntington’s chorea.9
While GABA itself has not been shown to be pharma-
cologically useful,10 many attempts have been made to
produce GABAergic drugs and prodrugs. Several
GABA receptor ligands derived from GABA (Fig. 1),
such as the selective agonists muscimol (7) and 4,5,6,7-
tetrahydroisoxazolo[5,4-c]-pyridine-3-ol (THIP, gabox-
adol, 8) and the antagonist gabazine (10), have been
developed over the years.4 In addition, the anticonvul-
sants Progabide (2), Vigabatrin (3) and Gabapentin
(4), containing a GABA substructure are marketed as
antiepileptic drugs.11
Also, c-butyrolactones and c-butyrolactams (Fig. 2)
are of biological relevance as GABA receptor ligands.12
c-Butyrolactones are common structural motifs encoun-
tered in a number of naturally occurring compounds
possessing therapeutic properties,13 and in particular c-
butyrolactones and their derivatives are GABAA modu-
lator agents of great interest14 because some of them
possess potent anticonvulsant activity in vivo.15 The c-
lactams, in turn (Fig. 2), have been shown to possess
anticonvulsant and antioxidant activity,16 gabapentin-
lactam (GBP-L) is neuroprotective in retinal ischae-
mia,17 and in addition, c-lactams can be useful as key
intermediates in the synthesis of pyrrolidines, the
biosynthetic precursors of GABA analogues.18
Here we describe the synthesis of sugar-fused b-disub-
stituted c-butyrolactones 21–22, (Scheme 1) c-butyro-
lactams 24–27 and a lipophilic b-disubstituted GABA
analogue 23 as potential GABA receptor ligands, where
the pharmacophore is engineered into the carbohydrate
scaffold in the form of a C-fructoside. A few examples of
sugar-fused GABA analogues on a galactose or glucose
*
Corresponding author. Tel.: +39 02 6448 3460; fax: +39 02 6448
3565; e-mail: laura.cipolla@unimib.it
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.03.013

A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
1841
OH
OH
OH
O
O
OH
O
HO
N
O
F
NH2
O
H2N
OH
O
HO
GABA (1)
RO
D-Fructose
OR
RO
Progabide (2)
21: R = Bn
22: R = H
Cl
NH2
OH
O
OH
OBn
19
OH
BnO
BnO
COOH
NHBoc
O
O
O
BnO
BnO
H2N
BnO
Vigabatrin (3)
Gabapentin (GBP, 4)
OBn
BnO
Cl
23
O
OH
O
OH
O
O
NH2
OBn
N
R1
O
RO
H2N
H2N
OR
GBP-analogue (6)
Baclofen (5)
RO
20
24: R = Bn, R1 = CO2tBu
25: R = Bn, R1 = H
HO
HO
N
NH
N
26: R = H, R1 = CO2tBu
27: R = R1 = H
NH2
O
O
THIP, Gaboxadol (8)
Muscimol (7)
HO
MeO
Scheme 1. Synthetic scheme towards GABA receptor ligands 21–27.
scaffold have been recently proposed,19 but no biological
evaluation of these compounds was reported.
Gabazine (10)
N
O
The fructose moiety acts as versatile scaffold,20 being
rich in stereochemistry and having a relatively rigid skel-
eton. For GABA receptor ligand action, penetration of
the blood-brain barrier (BBB) is required, and lipophil-
icity is the most important parameter that crucially
influences its penetration.21 It is worthy of note that
additional hydroxyl derivatization of the fructose scaf-
fold may be used to increase lipophilicity, as well as to
modulate the activity of pharmacophores or the recep-
tor specificity.
N
N
O
NH
4-PIOL (9)
NH2
NH
Figure 1. Chemical structure of some GABAergic ligands.
R2
R1
O
The synthesis of title compounds 21–27 requires
access to key intermediates 19 and 20. Although the syn-
thesis of compound 19 has been already described,22 the
synthesis of 20 was recently communicated without
experimental details in a preliminary account,23 and will
be therefore given here in detail. The anomeric position
and the hydroxyl group at C-1 of fructose were exploited
for the construction of the butyrolactone or lactam rings
with a spiro junction to the carbohydrate scaffold.
GABA lactones 21–22 were synthesized via key inter-
mediate 19, obtained from fully benzylated D-fructose
(Scheme 2), after C-allylation of the anomeric position.
C-Allyl fructofuranoside 19 was protected at the free
hydroxyl group affording the corresponding tert-butyl-
dimethylsilyl ether 28 with tert-butyldimethylchloro
silane and imidazole in dry dimethylformamide at reflux
(90% yield, Scheme 2).
R3
O
O
N
H
11: R1 = R2 = R3= H
GBP-L (15)
12: R1 = CH3, R2 = R3= H
13: R1 = R2 = CH3, R3= H
14: R1 = R3= CH3 R2 = H
O
NH
O
3,3-diethyl-2-pyrrolidinone (17)
NH
O
O
3-phenyl-2-pyrrolidinone (16)
N C
18
Figure 2. Chemical structure of some c-butyrolactones and c-butyro-
lactams as GABA receptor ligands.

1842
A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
3'
O
1
1'
2
COOH
OTBDMS
O
2
3
2'
7
6
O
O
6
5
5
O
1'
a
O
OTBDMS
b
c
OH
OBn
19
RO
BnO
BnO
1
BnO
4
3
4
OR
OBn
OBn
RO
BnO
BnO
BnO
28
29
21: R = Bn
22: R = H
d
Scheme 2. Reagents and conditions: (a) TBDMSCl, imidazole, dry DMF, reflux, 90%; (b) (i) 0.016 M OsO4 in t-BuOH, NaIO4, H2O–acetone–
t-BuOH 1:1:1, rt; (ii) 1.25 M NaH2PO4ꢀ2H2O, NaClO2, CH3CN, rt, 71% over 2 steps; (c) TFA–H2O 9:1, CH2Cl2, rt, 94%; (d) H2, Pd(OH)2–C, HCl
37%, MeOH–EtOAc 4:1, rt, 95%.
Oxidative cleavage of the double bond in the C-pro-
penyl appendage of compound 28 via a two-step
degradative oxidation by reaction with osmium
tetroxide–sodium periodate24 followed by treatment
group. In addition, the synthesis of these compounds
required the introduction of the amino functionality in
place of the hydroxyl group at C-1 of fructose. To this
end, key intermediate 20 was synthesized from alcohol
19 and subsequently transformed into title compounds
23–27 (Scheme 3).
Alcohol 19 was converted into aldehyde 30; the car-
bonyl group of 30 was transformed into the correspond-
ing amine required for the construction of the lactam
ring by oximation to compound 31 (NH2OHꢀHCl, pH
4.5, 92% yield as a mixture of E/Z isomers), followed
by reduction with LiAlH4 to amine 20 (quantitative
yield). The allyl-containing amine derivative 20 could
provide c-butyrolactam 24 by oxidation and c-lactam
formation.26 Hence, protection of amine 20 in the pre-
sence of Boc anhydride and triethylamine afforded
compound 32. Oxidative cyclization of 32 to the corres-
ponding lactam 24 proceeded in a two-step fashion
25
with NaH2PO4–NaClO2 (71% yield over two steps)
afforded acid 29, possessing the carboxylic function suit-
ably positioned for the formation of the c-butyrolactone
ring. Attempts to oxidize the double bond of 19 without
hydroxyl protection resulted in low yields in the desired
product. Acidic hydrolysis of the silyl ether with aque-
ous trifluoroacetic acid in dichloromethane directly
afforded benzylated c-lactone 21. Debenzylation by
hydrogenolysis using palladium hydroxide as a catalyst
afforded fully deprotected hydrophilic lactone 22 (95%
yield).
The synthesis of c-butyrolactams 24–27 and GABA
analogue 23 takes advantage once more of the C-pro-
penyl substituent for the introduction of the carboxylic
O
OH
O
NHR
OBn
O
c
b
BnO
d
N
OBn
a
19
CHO
OBn
30
BnO
BnO
BnO
BnO
BnO
20: R = H
32: R = Boc
31
e
OH
O
O
O
O
O
NBoc
OBn
f
g
NBoc
OBn
BnO
NH
BnO
h
HO
BnO
OH
BnO
j
HO
33
24
27
COOH
NHBoc
O
i
O
BnO
O
O
NBoc
OBn
23
BnO
HO
O
OH
26
NH
OBn
HO
BnO
25
BnO
˚
Scheme 3. Reagents and conditions: (a) PCC, CH2Cl2, 4 A m.s., 85% (Ref. 20); (b) NH2OHꢀHCl pH 4.5, MeOH–THF 1:1, 94%; (c) LiAlH4, THF,
quant. yield (d) (Boc)2, TEA, CH2Cl2, rt, 87%; (e) (i) 0.016M OsO4 in t-BuOH, NMO, THF–H2O 1:1; (ii) NaIO4, THF–H2O 1:1, rt, 85%; (f) PCC,
˚
4 A m.s., CH2Cl2, rt, 78%; (g) H2, Pd(OH)2–C, HCl 37%, MeOH–EtOAc 4:1, rt, 74%; (h) H2, Pd(OH)2–C, acetic acid, MeOH–EtOAc 4:1, rt, 70%; (i)
TFA–H2O 8:2, CH2Cl2, rt, 76%; (j) LiOH, THF, rt, 61%.

A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
1843
by initial osmylation–NaIO4 oxidation to lactol 33,
followed by further oxidation using pyridinium chloro-
chromate (PCC). Hydrolysis of lactam 24 using 1 N
lithium hydroxide provided the Boc-protected GABA
analogue 23 in 61% yield. Any attempt to obtain a fully
deprotected GABA analogue failed. Hydrogenolysis of
lactam 24 using palladium hydroxide as the catalysts
in the presence of hydrochloric acid afforded fully
deprotected and hydrophilic c-lactam 27 (74% yield);
on the contrary, hydrogenolysis with the same catalyst
but with acetic acid as the acid afforded N-Boc-
protected lactam 26 (70% yield). Finally, treatment of
lactam 25 with aqueous trifluoroacetic acid afforded
benzylated lactam 25 (76% yield).
Compounds 21–27 were characterized in receptor
binding studies at GABAA receptors using rat brain
membrane preparations. The ability of the new com-
pounds to bind to GABAA receptor was determined
using [3H]muscimol27 and the results are reported in
Table 1. Data show that compounds 21, 22, 24 and 26
possess affinity for GABAA receptor significantly
inhibiting [3H]muscimol binding in the lM range. In
particular, the most potent compound in the displace-
ment binding study is the N-Boc-protected lactam 26
with a 40% reduction of [3H]muscimol specific binding.
These biological data demonstrate that both butyro-
lactones 21–22, and N-substituted lactams 24 and 26,
are able to displace tritiated muscimol from the GABA
binding site. By this preliminary evaluation we can
argue that the sugar moiety does not seem to hinder
the binding, as both benzylated and deprotected
lactones and lactams had comparable activity. Hence,
the carbohydrate moiety can effectively be used to
modulate drug pharmacokinetic properties and lipophil-
icity. It is worth noting that the hydrophobic nature of
benzyl groups can facilitate BBB crossing, which is
one of the main issues to be addressed for CNS directed
drugs.
biological evaluation of title compounds as GABA
receptor ligands has been performed.
1. Experimental
1.1. Synthesis, general procedures
All solvents were dried over molecular sieves (Fluka),
for at least 24 h prior to use. When dry conditions were
required, the reactions were performed under an argon
atmosphere. Thin-layer chromatography (TLC) was
performed on Silica Gel 60 F254 plates (Merck) with
detection with UV light when possible, or charring with
a solution containing conc. H2SO4–EtOH–H2O in a
ratio of 5:45:45 followed by heating at 180 °C. Flash
column chromatography was performed on silica gel
230–400 mesh (Merck). The boiling range of petroleum
ether used as eluent in column chromatography is
40–60 °C. NMR spectra were recorded at 400 MHz
(1H) and at 100.57 MHz (13C) on a Varian MERCURY
instrument at 300 K. Chemical shift values (d) are
reported in ppm downfield from TMS as an internal
standard; J values are given in Hertz. Mass spectra were
recorded on a MALDI2 Kompact Kratos instrument,
with gentisic acid (DHB) as the matrix. Compounds
are numbered and named systematically as reported.19b
1.2. 1-Amino-2,5-Anhydro-3,4,6-tri-O-benzyl-1-deoxy-2-
C-(prop-2-enyl)-D-glucitol (20)
Oxime 31 (0.500 g, 1.02 mmol) was dissolved in dry
THF (5 mL), and 1.6 mL of 1 M LiAlH4 solution in
THF was added. The reaction mixture was stirred over-
night, then quenched by the addition of EtOAc; the pre-
cipitate was filtered off, and the solvent evaporated
under reduced pressure. Purification by flash chroma-
tography (6:4 EtOAc–EtOH), afforded 0.480 mg of 20,
as a colourless oil. 1H NMR (400 MHz, CDCl3): d
7.43–7.18 (m, 15H, Ph-H), 5.85–5.77 (m, 1H, H-20),
In summary, the synthesis of diverse butyrolactones
and lactams has been accomplished, and preliminary
5.13 (d, 1H, J3 a;2 ¼ 10:0 Hz, H-30a), 5.07 (d, 1H,
0
Table 1. Binding competition study of the synthesized products with
[3H] muscimol at GABAA receptor performed on rat cerebral
membranes
J3 b;2 ¼ 17:8 Hz, H-30b), 4.68–4.51 (m, 8H, PhCH2O,
0
NH2), 4.21 (dd, 1H, J4,5 = 7.0 Hz, J3,4 = 5.5 Hz, H-4),
4.09 (d, 1H, J3,4 = 5.5 Hz, H-3), 4.00 (dt, 1H,
J4,5 = 7.0 Hz, J5,6 = 4.0 Hz, H-5), 3.68 (dd, 1H,
J6a,6b = 10.6 Hz, J5,6a = 4.0 Hz, H-6a), 3.58 (dd, 1H,
J6a,6b = 10.6 Hz, J5,6b = 4.0 Hz, H-6b), 2.90 (d, 1H,
J1a,1b = 14.1 Hz, H-1a), 2.70 (d, 1H, J1a,1b = 14.1 Hz,
Compound
% [3H]Muscimol
specific bindinga
Significance
versus controlb
Control
21
22
100.00 3.053
75.55 1.013
77.84 0.446
99.33 2.073
70.77 4.391
76.75 0.029
60.42 6.668
96.80 1.976
P <0.01
P <0.05
n.s.
P <0.05
n.s.
23
24
0
0
0
0
H-1b), 2.39 (dd, 1H, J1 a;1 b ¼ 13:9 Hz; J1 a;2 7:1 Hz,
H-10a), 2.30 (dd, 1H, J1 a;1 b ¼ 13:9 Hz, 13.9 Hz,
0
0
25
26
J1 b;2 7:5 Hz, H-10b); 13C NMR (100.57 MHz, CDCl3):
d 138.20, 138.14, 138.12 (3s, Cq arom.), 133.49 (d,
C-20), 119.03 (t, C-30), 85.27 (s, C-2) 87.97, 84.66,
79.39 (3d, C-3, C-4, C-5), 73.74, 73.06, 72.94, 70.24
(4t, PhCH2O, C-6), 47.50 (t, C-1), 41.99 (t, C-10).
MALDI-MS m/z calcd for C30H35NO4: 473.60. Found:
0
0
P <0.05
n.s.
27
a Values are means SEM determined from at least three independent
experiments.
b Statistical analysis is performed with Kruskal–Wallis ANOVA for
nonparametric values followed by Dunns test.

1844
A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
475 [M+H]+, 497 [M+Na]+. Anal. Calcd for
C30H35NO4: C, 76.08; H, 7.45; N, 2.96. Found: C,
76.10; H, 7.48; N, 2.93.
MS m/z calcd for C8H12O6: 204.18. Found: 227
[M+Na]+, 243 [M+K]+. Anal. Calcd for C8H12O6: C,
47.06; H, 5.92. Found: C, 47.04; H, 5.97.
1.3. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-
(hydroxymethyl)-D-manno-heptonic acid lactone (21)
1.5. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-(amino-
methyl(N-tert-butoxycarbonyl))-D-manno-heptonic acid
(23)
To a solution of 29 (0.250 g, 0.412 mmol) in CH2Cl2
(8 mL), a mixture of 9:1 TFA–H2O (0.3 mL) was added
at 0 °C. The reaction mixture was allowed to warm at
room temperature and stirred for 1 h. The reaction
was quenched by slowly adding NaHCO3 until neutral
pH. The product was extracted with CH2Cl2 and the
organic layer was dried on sodium sulfate, filtered and
evaporated. Flash column chromatography of the resi-
due (8:2 petroleum ether–EtOAc) afforded 21 (0.184 g,
94%) as a dark yellow oil. 1H NMR (400 MHz, CDCl3):
d 7.40–7.22 (m, 15H, Ph-H), 4.63–4.51 (m, 6H,
To a solution of 24 (0.250 g, 0.436 mmol) in dry THF
(7 mL) 1 M LiOHꢀH2O (1.74 mL, 1.74 mmol) was
added, and the resulting solution was stirred at room
temperature for 2 h. THF was evaporated, and the
resulting residue was dissolved in Et2O and filtered.
The resulting filtrate was washed with Et2O and the
aqueous layer was acidified with 1 M HCl and extracted
with Et2O. The combined organic layers were dried
(Na2SO4), filtered and concentrated to dryness provid-
ing 23 as a yellow oil (0.157 g, 61%). 1H NMR
(400 MHz, CDCl3): d 7.25–7.16 (m, 15H, Ph-H), 5.05
(t, 1H, J = 6.3 Hz, NH), 4.53–4.33 (m, 6H, PhCH2O),
4.23 (d, 1H, J4,5 = 3.9 Hz, H-4), 4.11–4.07 (m, 1H,
H-6), 3.97 (dd, 1H, J5,6 = 6.8 Hz, J4,5 = 3.9 Hz, H-5),
3.47 (dd, 1H, J7a,7b = 10.3 Hz, J6,7a = 4.6 Hz, H-7a),
3.45–3.42 (m, 2H, H-10), 3.40 (dd, 1H, J7a,7b = 10.3 Hz,
J6,7b = 4.6 Hz, H-7b), 2.64 (d, 1H, J2a,2b = 14 Hz,
H-2a), 2.57 (d, 1H, J2a,2b = 14 Hz, H-2b), 1.48 (s, 9H,
C(CH3)3); 13C NMR (100.57 MHz, CDCl3): d 178.24
(s, CO-1), 156.76 (s, CO), 138.04, 137.83, 137.63 (3s,
Cq arom.), 86.63, 85.51, 83.89 (3d, C-4, C-5, C-6),
85.48, 80.48 (2s, C-3,C(CH3)3), 77.62, 77.30, 76.98,
69.99 (4t, PhCH2O, C-7), 44.20 (t, C-10), 40.42 (t, C-2),
28.65, 28.58, 27.98 (3q, C(CH3)3). MALDI-MS m/z
calcd for C34H41NO8: 591.69. Found: 615 [M+Na]+,
631 [M+K]+. Anal. Calcd for C34H41NO8: C, 69.02;
H, 6.98; N, 2.37. Found: C, 69.08; H, 6.95; N, 2.40.
PhCH2O), 4.39 (d, 1H, J1 a;1 b ¼ 9 Hz, H-10a), 4.37 (d,
0
0
1H, J1 a;1 b ¼ 9 Hz, H-10b), 4.23 (dt, 1H, J6,7 = 6.1 Hz,
J5,6 = 3 Hz, H-6), 4.04 (dd, 1H, J5,6 = 3 Hz,
J4,5 = 1.7 Hz, H-5), 3.94 (d, 1H, J4,5 = 1.7 Hz, H-4),
3.60 (dd, 1H, J7a,7b = 10 Hz, J6,7a = 5.5 Hz, H-7a),
3.52 (dd, 1H, J7a,7b = 10 Hz, J6,7b = 6.4 Hz, H-7b),
2.77 (d, 1H, J2a,2b = 18 Hz, H-2a), 2.63 (d, 1H,
J2a,2b = 18 Hz, H-2b); 13C NMR (100.57 MHz, CDCl3):
d 175.12 (s, CO), 138.02, 137.53, 137.09 (3s, Cq arom.),
87.36 (s, C-3), 85.81, 82.89, 82.77 (3d, C-4, C-5, C-6),
73.74 (t, C-10), 72.38, 72.18, 72.02, 70.42 (4t, PhCH2O,
C-7), 39.60 (t, C-2). MALDI-MS m/z calcd for
C29H30O6: 474.54. Found: 498 [M+Na]+, 514
[M+K]+. Anal. Calcd for C29H30O6: C, 73.40; H, 6.37.
Found: C, 73.38; H, 6.39.
0
0
1.4. 3,6-Anhydro-2-deoxy-3-C-(hydroxymethyl)-D-
manno-heptonic acid lactone (22)
1.6. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-(amino-
methyl(N-tert-butoxycarbonyl))-D-manno-heptonic acid
lactam (24)
Compound 21 (0.250 g, 0.527 mmol) was dissolved in 4:1
MeOH–EtOAc mixture (15 mL) and a few drops of HCl
37% and catalytic Pd(OH)2–C (10% w/w) were added.
The flask was purged 3 times with Ar and then filled with
H2. After 12 h, the catalyst was removed by filtration,
and the filtrate concentrated under reduced pressure.
The crude residue was purified by silica gel flash
chromatography (EtOAc) affording compound 22 as a
To a solution of 33 (0.250 g, 0.434 mmol) and molecular
˚
sieves (4 A, 300 mg) in dry CH2Cl2 (8 mL) pyridinium
chlorochromate (PCC, 0.187 g, 0.868 mmol) was added
and the mixture was stirred for 2 h. The resulting mix-
ture was filtered through a Celite pad eluting with
EtOAc. The filtrate was concentrated and purified by
silica gel flash chromatography (7:3 petroleum ether–
EtOAc) to give lactam 24 as a white solid (0.194 g,
78% yield). Mp = 82–85 °C; 1H NMR (400 MHz,
CDCl3): d 7.37–7.22 (m, 15H, Ph-H), 4.60–4.37 (m,
6H, PhCH2O), 4.2–4.15 (m, 1H, H-6), 4.02 (d, 1H,
1
yellow oil (0.102 g, 95%). H NMR (400 MHz, D2O): d
4.50 (d, 1H, J1 a;1 b ¼ 10:6 Hz, H-10a), 4.23 (d, 1H,
0
0
J1 a;1 b ¼ 10:6 Hz, H-10b), 4.07 (d, 1H, J4,5 = 3.8 Hz,
H-4), 3.89 (dd, 1H, J5,6 = 5.1 Hz, J4,5 = 3.8 Hz, H-5),
3.80 (dt, 1H, J5,6 = 5.1 Hz, J6,7 = 3.5 Hz, H-6), 3.60
(dd, 1H, J7a,7b = 12.3 Hz, J6,7a = 3.5 Hz, H-7a), 3.52
(dd, 1H, J7a,7b = 12.3 Hz, J6,7b = 5.3 Hz, H-7b), 2.81
(d, 1H, J2a,2b = 18 Hz, H-2a), 2.68 (d, 1H, J2a,2b = 18 Hz,
H-2b); 13C NMR (100.57 MHz, D2O): d 178.83 (s, CO),
87.47 (s, C-3), 83.69, 79.48, 76.64 (3d, C-4, C-5, C-6),
74.88 (t, C-10), 61.42 (t, C-7), 38.70 (t, C-2). MALDI-
0
0
J1 a;1 b ¼ 12:4 Hz, H-10a), 3.99 (dd, 1H, J5,6 = 3.2 Hz,
0
0
0
0
J4,5 = 1.8 Hz, H-5), 3.85 (d, 1H, J1 a;1 b ¼ 12:4 Hz,
H-10b), 3.82 (d, 1H, J4,5 = 1.8 Hz, H-4), 3.57 (dd, 1H,
J7a,7b = 10 Hz, J6,7a = 5.6 Hz, H-7a), 3.49 (dd, 1H,
J7a,7b = 10 Hz, J6,7b = 6.1 Hz, H-7b), 2.77 (d, 1H,

A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
1845
J1 a;1 b ¼ 12:4 Hz, H-10b), 3.56 (dd, 1H, J7a,7b = 12.4 Hz,
J6,7a = 5.3 Hz, H-7a), 3.5 (dd, 1H, J7a,7b = 12.4 Hz,
J6,7b = 5.4 Hz, H-7b), 2.77 (d, 1H, J2a,2b = 18 Hz,
H-2a), 2.62 (d, 1H, J2a,2b = 18 Hz, H-2b), 1.34 (s, 9H,
C(CH3)3); 13C NMR (100.57 MHz, D2O): d 178.19 (s,
CO-1), 153.52 (s, CO), 87.94, 85.3 (2s, C-3, C(CH3)3),
85.55, 82.38, 81.99 (3d, C-4, C-5, C-6), 63.90 (t, C-7),
56.27 (t, C-2), 45.68 (t, C-10), 28.94, 28.54, 27.85 (3q,
C(CH3)3). MALDI-MS m/z calcd for C13H21NO7:
303.31. Found: 326 [M+Na]+, 342 [M+K]+. Anal.
Calcd for C13H21NO7: C, 51.48; H, 6.98; N, 4.62.
Found: C, 51.53; H, 6.92; N, 4.65.
0
0
J2a,2b = 18 Hz, H-2a), 2.64 (d, 1H, J2a,2b = 18 Hz,
H-2b), 1.5 (s, 9H, C(CH3)3); 13C NMR (100.57 MHz,
CDCl3): d 171.70 (s, CO-1), 149.97 (s, CO), 138.12,
137.66, 137.34 (3s, Cq arom.), 85.9, 83.21, 82.23 (3d,
C-4, C-5, C-6), 83.2, 83.0 (2s, C-3,C(CH3)3), 73.60,
72.11, 71.95, 70.44 (4t, PhCH2O, C-7), 53.06 (t, C-2),
43.92 (t, C-10), 28.96, 28.27, 27.75 (3q, C(CH3)3). MAL-
DI-MS m/z calcd for C34H39NO7: 573.68. Found: 597
[M+Na]+, 613 [M+K]+. Anal. Calcd for C34H39NO7:
C, 71.18; H, 6.85; N, 2.44. Found: C, 71.20; H, 6.90;
N, 2.40.
1.7. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-(amino-
methyl)-D-manno-heptonic acid lactam (25)
1.9. 3,6-Anhydro-2-deoxy-3-C-(aminomethyl)-D-manno-
heptonic acid lactam (27)
A solution of 24 (0.250 g, 0.436 mmol) in dry CH2Cl2
(7 mL) and 80% aqueous TFA (13 mL) was stirred for
2 h at room temperature. Then, the mixture was neutral-
ized with an satd aq NaHCO3, and extracted with
CH2Cl2. The organic layer was dried (Na2SO4), filtered
and concentrated to dryness and then purified by silica
gel flash chromatography (5:5 petroleum ether–EtOAc)
to give 25 as a yellow oil (0.157 g, 76% yield). 1H
NMR (400 MHz, CDCl3): d 7.3–7.14 (m, 15H, Ph-H),
5.5 (s, 1H, NH), 4.52–4.31 (m, 6H, PhCH2O), 4.12–
4.10 (m, 1H, H-6), 3.91 (dd, 1H, J5,6 = 3.5 Hz,
J4,5 = 1.9 Hz, H-5), 3.8 (d, 1H, J4,5 = 1.9 Hz, H-4),
Compound 24 (0.250 g, 0.436 mmol) was dissolved in
4:1 MeOH–EtOAc mixture (15 mL) and a few drops
of HCl 37% and catalytic Pd(OH)2–C (10% w/w) were
added. The flask was purged 3 times with Ar and then
filled with H2. After 12 h, the catalyst was removed by
filtration, and the filtrate concentrated under reduced
pressure. The crude residue was purified by silica gel
flash chromatography (7.5:2:0.5 EtOAc–MeOH–H2O)
affording compound 27 as a yellow oil (0.088 g, 74%).
1H NMR (400 MHz, D2O): d 4.24 (s, 1H, NH), 3.99
(d, 1H, J4,5 = 4 Hz, H-4), 3.88 (dd, 1H, J5,6 = 5.3 Hz,
J4,5 = 4 Hz, H-5), 3.8–3.76 (m, 1H, H-6), 3.66 (d, 1H,
3.7 (d, 1H, J1 a;1 b ¼ 12 Hz, H-10a), 3.5 (dd, 1H,
J7a,7b = 10 Hz, J6,7a = 5.6 Hz, H-7a), 3.42 (dd, 1H,
J7a,7b = 10 Hz, J6,7b = 6.1 Hz, H-7b), 3.4 (d, 1H,
0
0
J1 a;1 b ¼ 12 Hz, H-10a), 3.6 (dd, 1H, J7a,7b = 12.3 Hz,
0
0
J6,7a = 3.6 Hz, H-7a), 3.51 (dd, 1H, J7a,7b = 12.3 Hz,
J1 a;1 b ¼ 12 Hz, H-10b), 2.52 (d, 1H, J2a,2b = 17.5 Hz,
H-2a), 2.44 (d, 1H, J2a,2b = 17.5 Hz, H-2b); 13C NMR
(100.57 MHz, CDCl3): d 175.4 (s, CO), 138.16, 137.74,
137.44 (3s, Cq arom.), 87.45 (s, C-3), 86.14, 83.41,
82.10 (3d, C-4, C-5, C-6), 73.59, 72.06, 71.89, 70.52
(4t, PhCH2O, C-7), 48.74 (t, C-2), 41.45 (t, C-10). MAL-
DI-MS m/z calcd for C29H31NO5: 473.56 Found: 497
[M+Na]+, 513 [M+K]+. Anal. Calcd for C29H31NO5:
C, 73.55; H, 6.60; N, 2.96. Found: C, 73.50; H, 6.64;
N, 2.99.
J6,7b = 5.4 Hz, H-7b), 3.24 (d, 1H, J1 a;1 b ¼ 12 Hz,
H-10b), 2.58 (d, 1H, J2a,2b = 18 Hz, H-2a), 2.42 (d, 1H,
J2a,2b = 18 Hz, H-2b); 13C NMR (100.57 MHz, D2O):
d 180.72 (s, CO), 89.85 (s, C-3), 85.58, 82.38, 79.20
(3d, C-4, C-5, C-6), 63.99 (t, C-7), 52.15 (t, C-2), 43.41
(t, C-10). MALDI-MS m/z calcd for C8H13NO5:
203.19. Found: 226 [M+Na]+, 242 [M+K]+. Anal.
Calcd for C8H13NO5: C, 47.29; H, 6.45; N, 6.89. Found:
C, 47.20; H, 6.40; N, 6.91.
0
0
0
0
1.10. 2,5-Anhydro-3,4,6-tri-O-benzyl-2-C-(prop-2-enyl)-
1-O-tert-butyldimethylsilyl-D-glucitol (28)
1.8. 3,6-Anhydro-2-deoxy-3-C-(aminomethyl(N-tert-
butoxycarbonyl))-D-manno-heptonic acid lactam (26)
To a solution of 19 (0.250 g, 0.527 mmol) in dry DMF
(10 mL), tert-butyldimethylsilyl chloride (TBDMSCl,
0.198 g, 1.32 mmol) and imidazole (0.106 g, 1.58 mmol)
were added under argon atmosphere, and the resulting
mixture was stirred under reflux for 12 h. The solvent
was then removed under reduced pressure and the
residue dissolved in CH2Cl2 and washed with brine.
The organic layer was dried (Na2SO4), filtered and
evaporated. The crude residue was purified by silica
gel flash chromatography (9:1 petroleum ether–EtOAc),
affording 28 as a yellow oil (0.279 g, 90% yield). 1H
NMR (400 MHz, CDCl3): d 7.38–7.25 (m, 15H,
Ph-H), 5.81–5.79 (m, 1H, H-20), 5.10 (d, 1H,
Compound 24 (0.250 g, 0.436 mmol) was dissolved in
4:1 MeOH–EtOAc mixture (15 mL) and a few drops
of acetic acid and catalytic Pd(OH)2–C (10% w/w) were
added. The flask was purged 3 times with Ar and then
filled with H2. After 12 h, the catalyst was removed by
filtration, and the filtrate concentrated under reduced
pressure. The crude residue was purified by silica gel
flash chromatography (9:1 EtOAc–MeOH) affording
1
compound 26 as a yellow oil (0.093 g, 70%). H NMR
(400 MHz, D2O): d 3.97 (d, 1H, J4,5 = 4.4 Hz, H-4),
3.93 (d, 1H, J1 a;1 b ¼ 12:4 Hz, H-10a), 3.88 (m, 1H,
0
0
H-5), 3.8–3.76 (m, 1H, H-6), 3.66 (d, 1H,

1846
A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
J3 a;2 ¼ 10:2 Hz, H-30a), 4.98 (d, 1H, J3 b;2 ¼ 17 Hz,
H-30b), 4.75–4.46 (m, 6H, PhCH2O), 4.10 (dq, 1H,
646 [M+K]+. Anal. Calcd for C35H46O7Si: C, 69.27;
H, 7.64; Si, 4.63. Found: C, 69.29; H, 7.60; Si, 4.60.
0
0
J4,5 = 5.3 Hz, J5,6 = 1.3 Hz, H-5), 4.04 (dd, 1H, J4,5
=
5.3 Hz, J3,4 = 3.6 Hz, H-4), 3.96 (d, 1H, J3,4 = 3.6 Hz,
H-3), 3.69 (dd, 1H, J6a,6b = 10.0 Hz, J5,6a = 1.4 Hz,
H-6a), 3.63 (dd, 1H, J6a,6b = 10.0 Hz, J5,6b = 1.2 Hz,
H-6b), 3.60–3.52 (m, 2H, H-1), 2.52–2.41 (m, 2H,
H-10), 0.88 (s, 9H, C(CH3)3), 0.04 (s, 3H, SiCH3),
ꢁ0.08 (s, 3H, SiCH3); 13C NMR (100.57 MHz, CDCl3):
d 138.27, 138.16, 137.69 (3s, Cq arom.), 133.05 (d, C-20),
119.18 (t, C-30), 84.40 (s, C-2), 86.69, 83.58, 79.46 (3d,
C-3, C-4, C-5), 73.74, 73.25, 70.96, 69.72 (4t, PhCH2O,
C-6), 66.51 (t, C-1), 40.45 (t, C-10), 26.94, 25.17, 24.53
(3q, C(CH3)3), 20.15 (s, C(CH3)3), ꢁ4.6, ꢁ4.8 (2q,
SiCH3). MALDI-MS m/z calcd for C36H48O5Si:
588.33. Found: 611 [M+Na]+, 627 [M+K]+. Anal.
Calcd for C36H48O5Si: C, 73.43; H, 8.22; Si, 4.77.
Found: C, 73.46; H, 8.20; Si, 4.73.
1.12. 2,5-Anhydro-3,4,6-tri-O-benzyl-2-C-(prop-2-enyl)-
D-glucose oxime (31)
Aldehyde 30 (0.570 g, 1.21 mmol) was dissolved in a 1:1
THF–EtOH mixture (5 mL), and 0.252 g of hydroxyl-
amine hydrochloride (3.63 mmol) dissolved in a pH 4.5
acetate buffer (2.5 mL) was added. After 4 h, the reac-
tion mixture was extracted with dichloromethane, the
organic layer dried over Na2SO4, filtered and evapo-
rated to dryness. The crude was purified by flash
chromatography (8.5:1.5 petroleum ether–EtOAc),
affording 0.545 g of 31, as a colourless oil (92% yield).
1H NMR (400 MHz, CDCl3): d 7.51 (s, 1H, OH),
7.38–7.22 (m, 16H, Ph-H, H-1), 5.95–5.82 (m, 1H,
H-20), 5.13 (d, 1H, J3 a;2 ¼ 10:2 Hz, H-30a), 5.09 (d, 1H,
0
0
J3 b;2 ¼ 17:0 Hz, H-30b), 4.59–4.49 (m, 6H, PhCH2O),
0
1.11. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-
(hydroxymethyl(O-tert-butyldimethylsilyl))-D-manno-
heptonic acid (29)
4.16 (q, 1H, J4,5 = J5,6a = J5,6b 4.8 Hz, H-5), 4.14–4.09
(m, 2H, H-3, H-4), 3.69–3.52 (m, 2H, H-6), 2.66 (bd,
2H, H-10); 13C NMR (100.57 MHz, CDCl3): d 152.14
(d, C-1), 138.25, 137.95, 137.71 (3s, Cq arom.), 133.00
(d, C-20), 119.31 (t, C-30), 84.20 (s, C-2) 87.48, 84.72,
81.25 (3d, C-3, C-4, C-5), 73.77, 72.71, 72.55, 70.67
(4t, PhCH2O, C-6), 40.11 (t, C-10). MALDI-MS m/z
calcd for C30H33NO5: 487.59. Found: 489 [M+H]+,
511 [M+Na]+, 527 [M+K]+. Anal. Calcd for
C30H33NO5: C 73.90; H 6.82; N 2.87. Found: C, 73.88;
H, 6.80; N, 2.89.
To a solution of 28 (0.250 g, 0.424 mmol) in 1:1:1 H2O–
acetone-t-BuOH (10 mL), 0.016M OsO4 in t-BuOH
(1.3 mL, 0.021 mmol) was added. After 30 min the mix-
ture was treated with sodium periodate (0.181 g,
0.848 mmol) and 2 h later the reaction solution was fil-
tered and the solvents evaporated in vacuo without heat-
ing, affording the desired aldehyde that was used
without further purification. To a solution of the alde-
hyde (0.250 g, 0.423 mmol) in dry CH3CN (8 mL) a
solution of NaH2PO4ꢀ2H2O 1.25 M (3.3 mL,
4.23 mmol) and sodium chlorite (0.382 g, 4.23 mmol)
was added at rt. After 3 h, the solvents were evaporated
in vacuo and the resulting residue was dissolved in
CH2Cl2, filtered and evaporated. The residue was puri-
fied by flash column chromatography (8:2 petroleum
ether–EtOAc) affording 29 as a yellow oil (0.182 g,
1.13. 1-Amino-2,5-Anhydro-3,4,6-tri-O-benzyl-1-deoxy-
2-C-(prop-2-enyl)-N-tert-butoxycarbonyl-D-glucitol (32)
To a solution of 20 (0.250 g, 0.528 mmol) and Et3N
(0.223 mL, 1.58 mmol) in dry CH2Cl2 (7 mL) Boc anhy-
dride (0.138 g, 0.634 mmol) was added at 0 °C. Then the
mixture was warmed to room temperature and stirred at
rt overnight. Volatiles were removed, and the residue
was dissolved in EtOAc and washed with H2O. The
organic layer was dried (Na2SO4), filtered, concentrated
to dryness and then purified by silica gel flash
chromatography (9:1 petroleum ether–EtOAc) to give
the desired product 32 as a yellow oil (0.264 g, 87%
yield). 1H NMR (400 MHz, CDCl3): d 7.38–7.24 (m,
15H, Ph-H), 5.89–5.79 (m, 1H, H-20), 5.37 (t, 1H,
JNH,1a = JNH,1b = 5.6 Hz, NH), 5.13 (dd, 1H,
1
71% yield). H NMR (400 MHz, CDCl3): d 7.30–7.19
(m, 15H, Ph-H), 4.54–4.45 (m, 6H, PhCH2O), 4.28–
4.26 (m, 1H, H-6), 3.98 (dd, 1H, J5,6 = 5.6 Hz,
J4,5 = 2.2 Hz, H-5), 3.96 (d, 1H, J4,5 = 2.2 Hz, H-4),
3.84 (d, 1H, J1 a;1 b ¼ 10:3 Hz, H-10a), 3.73 (d, 1H,
0
0
J1 a;1 b ¼ 10:3 Hz, H-10b), 3.56 (dd, 1H, J7a,7b = 9.8 Hz,
J6,7a = 6.0 Hz, H-7a), 3.46 (dd, 1H, J7a,7b = 9.8 Hz,
J6,7b = 6.1 Hz, H-7b), 2.92 (d, 1H, J2a,2b = 15 Hz,
H-2a), 2.74 (d, 1H, J2a,2b = 15 Hz, H-2b), 0.86 (s, 9H,
C(CH3)3), 0.03 (s, 3H, SiCH3), ꢁ0.02 (s, 3H, SiCH3);
13C NMR (100.57 MHz, CDCl3): d 173.5 (s, CO),
138.77, 138.68, 137.91 (3s, Cq arom.), 83.95 (s, C-3),
86.19, 82.45, 78.93 (3d, C-4, C-5, C-6), 73.17, 72.95,
72.07, 70.25 (4t, PhCH2O, C-7), 63.43 (t, C-10), 36.50
(t, C-2), 26.21, 25.95, 23.43 (3q, C(CH3)3), 21.25 (s,
C(CH3)3), ꢁ3.9, ꢁ4.2 (2q, SiCH3). MALDI-MS m/z
calcd for C35H46O7Si: 606.82. Found: 630 [M+Na]+,
0
0
J3 a;2 ¼ 10:2 Hz, J3 a;2 ¼ 2:1 Hz, H-30a), 5.06 (dd, 1H,
0
0
J3 b;2 ¼ 17:1 Hz, J3 a;2 ¼ 2:1 Hz, H-30b), 4.63–4.47 (m,
0
0
6H, PhCH2O), 4.20 (dd, 1H, J4,5 = 6.7 Hz, J3,4
=
5.4 Hz, H-4), 4.09 (d, 1H, J3,4 = 5.4 Hz, H-3), 3.99 (m,
1H, H-5), 3.61 (dd, 1H, J6a,6b = 10.5 Hz, J5,6a
=
4.0 Hz, H-6a), 3.49 (dd, 1H, J6a,6b = 10.5 Hz,
J5,6b = 4.0 Hz, H-6b), 3.38 (m, 2H, H-1), 2.43 (dd, 1H,
J1 a;1 b ¼ 14:2 Hz, J1 a;2 ¼ 7:0 Hz, H-10a), 2.34 (dd, 1H,
0
0
0
0
J1 a;1 b ¼ 14:2 Hz, J1 b;2 ¼ 7:6 Hz, H-10b), 1.45 (s, 9H,
0
0
0
0

A. C. Arau´jo et al. / Carbohydrate Research 343 (2008) 1840–1848
1847
C(CH3)3); 13C NMR (100.57 MHz, CDCl3): d 161.74 (s,
CO), 138.45, 138.34, 138.22 (3s, Cq arom.), 132.89 (d,
C-20), 119.94 (t, C-30), 88.17, 85.18, 79.74 (3d, C-3,
C-4, C-5), 85.67, 85.11 (2s, C-2,C(CH3)3), 73.54, 73.09,
72.59, 69.64 (4t, PhCH2O, C-6), 40.85 (t, C-1), 38.46
(t, C-10), 28.20, 26.7, 25.6 (3q, C(CH3)3). MALDI-MS
m/z calcd for C35H43NO6: 573.72. Found: 597 [M+Na]+,
613 [M+K]+. Anal. Calcd for C35H43NO6: C, 73.27;
H, 7.55; N, 2.44. Found: C, 73.31; H, 7.50; N, 2.54.
by Frosini et al.27 with slight modifications. The whole
brain was homogenized in 10 vol of cold 0.32 M sucrose
and the homogenate was centrifuged at 2500g for 10 min
at 4 °C. The supernatant was then centrifuged at 48,000g
for 30 min at 4 °C and the pellet (crude synaptic mem-
branes) was resuspended in Tris–HCl 50 mM, pH 7.4
(1 mL/1 g wet weight) and frozen (ꢁ20 °C) for 24 h.
Thawed membranes were then resuspended in Tris–
HCl containing Triton X-100 (0.05% w/v) to obtain a
final protein concentration of 1 mg/mL. The mixture
was incubated for 30 min at 37 °C and then centrifuged
at 48,000g for 20 min at 4 °C. The pellet was then
washed three times with Tris–HCl and frozen at
ꢁ20 °C before use. The pellet, resuspended in Tris–
HCl to obtain a final protein concentration of 2 mg/mL,
was used for displacement binding studies. [3H]Musci-
mol (10 nM) served as radioligand and nonspecific
binding was determined in the presence of nonlabelled
muscimol (500 lM) and represented about 30–40% of
the total binding. Compounds 22, 26 and 27 were
diluted in Tris–HCl 50 mM pH 7.4, to obtain the final
concentration of 500 lM, whereas stock solutions of
compounds 21, 24 and 25 were prepared in ethanol
and then diluted in Tris–HCl to obtain the final concen-
tration of 500 lM. The binding reaction consisted in
0.1 mg of membranes incubated with the radioligand
in the presence or absence of the test compounds and
was stopped after 30 min at 4 °C by rapid filtration
under vacuum through glass-fibre filter (GO/B, What-
man). The filters were washed twice with 2 mL portions
of ice-cold Tris–HCl buffer and then dissolved in 8 mL
of scintillation fluid (UltimaGold). Filter-bound radio-
activity was determined by a scintillation counter. For
all compounds three independent experiments were
performed in triplicate. Results are expressed as percent-
age of control [3H]Muscimol specific binding and
analyzed by Graph Pad Prism, using the Kruskal–Wallis
ANOVA for nonparametric data followed by Dunns
test for specific comparisons.
1.14. 3,6-Anhydro-4,5,7-tri-O-benzyl-2-deoxy-3-C-(ami-
nomethyl(N-tert-butoxycarbonyl))-D-manno-heptonic
acid lactol (33)
To a solution of 32 (0.250 g, 0.436 mmol) in dry THF
(6 mL) 4-methylmorpholine N-oxide (NMO, 0.147 g,
1.09 mmol) and 0.016 M OsO4 in t-BuOH (1.38 mL,
0.022 mmol) were added at 0 °C, and the resulting mix-
ture was stirred at room temperature for 5 h. Additional
NMO (0.071 g) was added, and stirring was continued
overnight. The mixture was poured into 1 M Na2S2O3
and extracted with EtOAc. The organic layer was dried
(Na2SO4), filtered, concentrated to dryness to give the
diol intermediate that was used without further purifica-
tion. To a solution of the diol in THF (5 mL) aqueous
solution of NaIO4 (158 mg, 0.741 mmol in 1 mL) was
added at 0 °C, and the mixture was stirred for 3 h,
poured into ice water and extracted with EtOAc. The
organic layer was dried (Na2SO4), filtered, concentrated
to dryness and then purified by silica gel flash chromato-
graphy (7:3 petroleum ether–EtOAc) to give lactol 33 as
1
a yellow oil (0.213 g, 85% yield). H NMR (400 MHz,
CDCl3): d 7.28–7.12 (m, 15H, Ph-H), 5.37 (t, 1H,
J1,2 = 6 Hz, H-1), 4.51–4.30 (m, 6H, PhCH2O), 4.10–
4.02 (m, 1H, H-6), 4.0 (d, 1H, J4,5 = 1.6 Hz, H-4), 3.85
(dd, 1H, J5,6 = 3.5 Hz, J4,5 = 1.6 Hz, H-5), 3.81 (d,
1H, J1 a;1 b ¼ 12 Hz, H-10a), 3.51 (dd, 1H, J7a,7b = 17 Hz,
0
0
J6,7a = 5.8 Hz, H-7a), 3.40 (dd, 1H, J7a,7b = 17 Hz,
0
0
J6,7b = 7 Hz, H-7b), 3.37 (d, 1H, J1 a;1 b ¼ 12 Hz,
H-10b), 2.2 (dd, 1H, J2a,2b = 14 Hz, J1,2a = 5.2 Hz, H-2a),
1.9 (dd, 1H, J2a,2b = 14 Hz, J1,2b = 6.8 Hz, H-2b), 1.15
(s, 9H, C(CC(CH3)3)); 13C NMR (100.57 MHz, CDCl3):
d 151.15 (s, CO), 138.54, 138.78, 137.25 (3s, Cq arom.),
85.87, 83.54, 82.31 (3d, C-4, C-5, C-6), 83.60, 83.38 (2s,
C-3,C(CH3)3), 73.55, 72.63, 71.97, 70.50 (4t, PhCH2O,
C-7), 54.43 (t, C-10), 39.54 (t, C-2), 28.94, 28.35, 27.80
(3q, C(CH3)3). MALDI-MS m/z calcd for C34H41NO7:
575.69. Found: 599 [M+Na]+, 615 [M+K]+. Anal.
Calcd for C34H41NO7: C, 70.93; H, 7.18; N, 2.43.
Found: C, 71.00; H, 7.20; N, 2.40.
Acknowledgements
`
This work was supported by Ministero dell’Universita e
della Ricerca under Contract MIUR-PRIN 2005
n.2005037725 and Fundaßcao para a Cieˆncia e Tecnolo-
˜
gia (FCT) SFRH/BD/17815/2004; we gratefully
acknowledge Dr. Pietro Fumagalli for his technical
support for GABAA receptor binding assays.
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