Facile synthesis of oxo-/thioxopyrimidines and tetrazoles C-C linked to sugars as novel non-toxic antioxidant acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.carres.2011.11.006

Microwave-assisted synthesis of oxo-/thioxopyrimidines and tetrazoles linked to furanoses with d-xylo and d-ribo configuration, and to a d-galacto pyranose is reported and compared to conventional methods. Reaction of dialdofuranoses and dialdopyranoses w

Full text of DOI:10.1016/j.carres.2011.11.006

Carbohydrate Research 347 (2012) 47–54
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Facile synthesis of oxo-/thioxopyrimidines and tetrazoles C–C linked to sugars
as novel non-toxic antioxidant acetylcholinesterase inhibitors
J.A. Figueiredo a, M.I. Ismael a, , J.M. Pinheiro a, A.M.S. Silva b, J. Justino c, F.V.M. Silva c, M. Goulart c,
⇑
D. Mira c, M.E.M. Araújo d, R. Campoy d, A.P. Rauter d
a Departamento de Química, Unidade I&D Materiais Têxteis e Papeleiros da Universidade da Beira Interior, Av. Marquês d’Ávila e Bolama, 6201-001 Covilhã, Portugal
b Departamento de Química & QOPNA, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
c Escola Superior Agrária, Instituto Politécnico de Santarém, Complexo Andaluz, 2001-904 Santarém, Portugal
d Universidade de Lisboa, Faculdade de Ciências, Centro de Química e Bioquímica/Departamento de Química e Bioquímica, Carbohydrate Chemistry Group, Ed. C8,
5° Piso, Campo Grande, 1749-016 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2011
Received in revised form 30 October 2011
Accepted 4 November 2011
Available online 13 November 2011
Microwave-assisted synthesis of oxo-/thioxopyrimidines and tetrazoles linked to furanoses with D-xylo
and D-ribo configuration, and to a D-galacto pyranose is reported and compared to conventional methods.
Reaction of dialdofuranoses and dialdopyranoses with a b-keto ester and urea or thiourea under micro-
wave irradiation at 300 W gave in 10 min the target molecules containing the 2-oxo- or 2-thioxo-pyrim-
idine ring in high yield. The tetrazole-derived compounds were obtained in two steps by reaction of the
formyl group with hydroxylamine hydrochloride, copper sulfate, triethylamine and dicyclohexylcarbodi-
imide to give an intermediate nitrile, which was then treated with sodium azide. The use of microwave
irradiation in the latter step also resulted in a considerably shorter reaction time (10 min) compared to
hours under conventional heating to obtain a complete starting materials conversion. Acetylcholinester-
Keywords:
Sugar-linked heterocycles
Oxo-/thioxopyrimidine
Tetrazole
Acetylcholinesterase inhibitors
Antioxidant activity
ase inhibition ranged from 20% to 80% for compounds concentration of 100 lg/mL, demonstrating the
potential of this family of compounds for the control of Alzheimer’s disease symptoms. Most of the com-
pounds showed antioxidant activity in the b-carotene/linoleic acid assay, some of them exhibiting IC50
values in the same order of magnitude as those of gallic acid. The bioactive compounds did not show
cytotoxic effects to human lymphocytes using the MTT method adapted for non-adherent cells, nor geno-
toxicity determined by the short-term in vitro chromosomal aberration assay.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
heterocycles C–C linked to a non-anomeric position are known for
a variety of biological activities as pharmaceutical and agriculture
Alzheimer’s disease (AD) is a progressive dementia in which
memory and other cognitive functions, such as language and gen-
eral intellectual performance, also become impaired. It is one of the
most common age-related neurodegenerative diseases, considered
an urgent public health problem. Although the primary cause of AD
is still debated,1 research on its molecular pathogenesis has pro-
vided a robust framework to guide the search for effective new
pharmacological treatments, which is one of the major challenges
facing modern medicine. Acetylcholinesterase (AChE) inhibitors
were the first group of compounds that showed some promise in
the treatment of AD and have been effective for the control of mild
to moderate AD.2,3
applied products.5–8 As part of our ongoing search for bioactive com-
pounds derived from sugars, we have reported the synthesis and
bioactivity of pseudo-C-nucleosides with the heterocycles pyra-
zole,9 triazole9 and thiazole4 C–C linked to furanoses.4,9 We now
report on the synthesis of related compounds-type with the oxo-
and thioxopyrimidine rings synthesized by Biginelli reaction,10
which has been extended for the preparation of a large number of
oxopyrimidines11 starting from b-ketoesters or b-diketones, arylal-
dehyde and (thio)urea under photochemical12 or microwave irradi-
ation.13,14 These compounds have been investigated by a large
number of research groups due to their biological properties,
namely as antibacterial or antifungal agents,15 and as HIV inhibi-
tors16,17 of gp-120-CD4.18 They are active against cancer,19 and pres-
ent antihypertensive and anti-inflammatory activities.19 Hence, an
easy access to this new class of compounds embodying these het-
erocycles C–C linked to sugar residues is of high importance.
The medicinal, biochemical and pharmacological properties of
tetrazole compounds support a vast progress in their chemistry
Preliminary reports on compounds with a thiazolidinone ring
C–-C linked to the 40 position of a sugar residue showed that they
also act as cholinesterase inhibitors.4 Compounds possessing
⇑
Corresponding author.
E-mail address: iismael@ubi.pt (M.I. Ismael).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.006

48
J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
over the last years.20–22 Tetrazoles have a wide variety of applica-
tions as pharmaceutical products and explosives, in photography,
in information recording systems and as precursors of a variety
of nitrogen heterocycles.23–25 5-Substituted 1,2,3,4-tetrazoles are
described to possess antibacterial,26 antifungal,27 antiviral,28 anal-
gesic,29 anti-inflammatory,30 antiulcer31,32 and antihypertensive33
properties. It is known that the tetrazole ring can serve as an isos-
teric substitute for the carboxylic group in biologically active mol-
ecules, since both groups possess comparable acidity and size. The
tetrazole unit, however, has proven to be superior in resisting met-
abolic degradation.34–37 Synthesis of tetrazoles C–C linked to sug-
ars is also accomplished in this work and the target compounds
are evaluated regarding their ability to inhibit acetylcholinester-
ase. Oxidative stress is associated with Alzheimer’s and vascular
dementias, as well as with diabetes type II, among other patholo-
gies.38 Hence, evaluation of the antioxidant activity39 of the new
compounds by the b-carotene/linoleic acid assay is also reported.
Table 1
Yields obtained by methods A, B and Ca (for compounds 5–10) and D, E and Fb (for
compounds 15–18)
Compound number
Yield (%)
Conventional heating
Microwave irradiation
Method C
Method A
Method B
5
6
7
8
9
10
77
79
—
—
—
—
—
82
78
80
79
80
80
84
80
81
81
—
Method D
Method E
Microwave irradiation
Method F
15
16
17
18
96
99
97
98
98
98
99
99
98
98
99
99
a
Method A: 75 °C, 5 h; Method B: 75 °C, 5 h and 72 h, room temp; Method C:
2. Results and discussion
2.1. Chemistry
MW (300 W), 10 min.
Method D: NaN3, NH4Cl, DMF, 120 °C, 3 h; Method E: NaN3, ZnCl2, H2O, 100 °C,
2 h 30 min; Method F: NaN3, ZnCl2, H2O, MW (300 W), 10 min.
b
2.1.1. Synthesis of 4-glycosyl-2-oxo-/2-thioxopyrimidines
Reaction of dialdofuranoses 1–3 with ethyl acetoacetate and
urea/thiourea was accomplished under conventional heating or
microwave irradiation giving the 2-oxopyrimidines 5–7 and the
2-thioxopyrimidines 8–10 (Scheme 1, Table 1), in high yield. Con-
ventional heating required 5 h for reaction completion with xylo-
furanosyl-heterocycles, while the ribofuranosyl and galactosyl
analogues required 5 h at 75 °C and additional stirring for 72 h at
room temperature. However the microwave-assisted reactions
were completed within 10 min in the presence of 10 times smaller
volume of solvent than that used in the method described in the
literature,40 giving slightly higher yields.
The first reaction step consists of condensation of the aldehyde
with urea to generate an iminium intermediate which acts as an
electrophile for the nucleophilic addition of the keto enol ester,
and the ketone carbonyl of the resulting adduct undergoes conden-
sation with NH2 to give the cyclized product. The formation of a
single reaction product may result from the exocyclic attack of
the keto enol ester to the iminium species which seems to be sta-
bilized by a hydrogen bond between the free amino group and the
endocyclic ring oxygen. The formation of one single diastereoiso-
mer with assigned (R)-configuration for the new stereogenic cen-
ter, also confirms the proposed mechanism, in alternative to the
described acid-promoted enolization and aldol addition, followed
by addition of urea to the formed conjugated system, as described
by Li.41 These results are in full agreement with data reported in
the literature for similar reactions.40
The oxopyrimidine ring in compound 5 was confirmed by the
singlet at d 8.92 (NH-1), the doublet at d 5.66 (NH-3), the doublet
at d 4.57 (H-4), the signals of the ethyl carboxylate group at d 4.11
(quartet, CH2) and 1.18 (triplet, CH3), in addition to the singlet of
the methyl group CH3-6 at d 2.26. In the 13C NMR spectrum of 5
the ester carbonyl group appeared at d165.8, while the quaternary
carbons C-2, C-6 and C-5 appeared at d 155.0, 149.3, and 97.0,
respectively. The chemical shift of C-4 is assigned at d 51.42 and
the signal of the methyl group linked to C-6 emerged at d 18.41.
NMR spectra of compounds 6 and 7 exhibited a similar pattern re-
lated to the presence of this heterocycle, while compounds 8–10
showed the signal of C-2, which belongs to a thiocarbonyl group,
at higher field (d 176.6–174.6).
X
HN
CH3COCH2CO2Et, NH2CXNH2
R
CHO
R
NH
EtOH
(a)
1-3 R=S1, S2, S3; R'=OBn
4 R=S1, R'=H
EtO2C
CH3
2.1.2. Synthesis of 4-glycosyltetrazoles
5-7 X = O
(b)
8-10 X = S
The tetrazole ring was formed by the pathway presented in
Scheme 1. Sugar nitriles 11–14 were obtained by the reaction of
appropriate aldehydes with hydroxylamine hydrochloride, trieth-
ylamine, copper(II) sulfate pentahydrate, and the dehydration
agent DCC,9 using dichloromethane as solvent, at room tempera-
ture for 2 h. After workup and purification by flash chromatogra-
phy, compounds 11–14 were obtained in 95–97% yield. The IR
band at 2257–2259 cmꢀ1, and the 13C NMR signals at d 114–115
confirmed the formation of compounds 11–14. Reaction of the ni-
triles 11–14 with sodium azide, ammonium chloride in DMF at
120 °C for 3 h (method D) led to the formation of tetrazoles 15–
18 in 96–99% yield. The reaction conditions using sodium azide,
ZnCl2, water as solvent, at 100 °C for 2.5 h (method E) gave com-
pounds 15–18 in similar yields (97–98%). However, when these re-
agents were submitted to microwave irradiation (method F), the
tetrazoles were formed in 10 min and isolated in 98–99% yield.
H-40 resonated at d 5.25–5.74, shifted downfield to the correspond-
ing proton of the nitrile precursor, and the presence of the C@N
H
N
N
N
NaN3
(c)
R
R
CN
N
11 R=S1, R'=Bn
12 R=S2
15 R=S1, R'=Bn
13 R=S3
16 R=S2
14 R=S1, R'=H
17 R=S3
18 R=S1, R'=H
O
O
O
O
R =
OR'
O
O
O
O
O
O
O
OBn
S2
S3
S1
Scheme 1. (a) Method A: 75 °C, 5 h; Method B: 75 °C, 5 h and 72 h, rt; Method C:
MW (300 W), 10 min; (b) NH2OHꢁHCl, Pyr, H2O; CuSO4ꢁ5H2O, Et3N, DCC, CH2Cl2, 2 h,
rt; (c) Method D: NaN3, NH4Cl, DMF,120 °C, 3 h; Method E: NaN3, ZnCl2, H2O, 100 °C,
2 h 30 min; Method F: NaN3, ZnCl2, H2O, MW (100 W), 10 min.

J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
49
signal at d 152–156 confirmed the expected structure for com-
pounds 15–18.
Table 1 summarizes compounds’ yields synthesized either by
conventional heating or by microwave irradiation and illustrates
the advantages of the latter for the synthesis of this family of
compounds.
2.2. Biological activity
2.2.1. Inhibition of acethylcholinesterase activity
The inhibitory activity of the new synthetic compounds against
AChE (acetylcholinesterase), from bovine erythrocytes, was evalu-
ated using the Ellman’s spectrophotometric method42 to determine
the rate of hydrolysis of acetylthiocholine in the presence of the
inhibitor. The generated thiocholine reacts with 5,50-dithiobis-2-
nitrobenzoic acid (DTNB) to produce a yellow compound that is de-
tected spectrophotometrically (k = 410 nm). The enzyme activity
(%) and the enzyme inhibition (%) were calculated from the change
Figure 2. Antioxidant activity of compounds 5–8, 10, 15 and 18, evaluated by the b-
carotene/linoleic acid bleaching test.
of absorbance with time (V =
DAbs/Dt) as follows:
Enzyme activityð%Þ ¼ 100 ꢂ V=Vmax
Enzyme inhibitionð%Þ ¼ 100 ꢀ Enzyme activityð%Þ
Maximum rate (Vmax) is obtained when no inhibitor is used
while V is the rate obtained in the presence of the inhibitor.
The most promising compounds 8 and 9 have the thioxopyrim-
idine ring in their structure linked to the furanose moiety and pro-
moted 69% and 83% enzyme inhibition ( Fig. 1). All other
compounds gave inhibition lower than 54% with the exception of
the tetrazole 18, which also led to
a considerable enzyme
inhibition.
Figure 3. Cell proliferation after exposure to compounds 7, 8, 10, 16, and 18.
2.2.2. b-Carotene/linoleic acid bleaching test
The antioxidant activity of compounds 5–10 and 15–18 was
evaluated by the b-carotene bleaching assay and the results ob-
tained are presented in Figure 2. This test is appropriate for lipo-
philic compounds and measures their ability to inhibit lipid
peroxidation. For comparative purposes the antioxidant activity
of two standards, gallic acid (IC50 = 0.833 0.044 mg/mL) of natu-
ral origin and the synthetic antioxidant BHT (butylated hydroxy-
toluene) (IC50 = 0.0045 0.0008 mg/mL), was also evaluated. The
latter has been used in the food industry but doubts about its
safety for human health led to the investigation of new antioxi-
dants. Compounds 9, 16 and 17 presented IC50 P 1.5 mg/mL and
idant activity, when compared to BHT and also to gallic acid, which
is a polar antioxidant.
2.2.3. Toxicity studies
Acute toxicity assessment was performed by the MTT method45
adapted for non-adherent cells.46
As shown in Figure 3, only compound 8 has a mitotic index
indicative of toxicity. All other compounds, including the most ac-
tive ones 9 and 18, do not present any toxicity. Viability of the cells
was not affected (Fig. 4) and no evidence of genotoxic risk was ob-
served for all the bioactive compounds.
were considered not active. However compounds
8
(IC50 = 0.103 0.002 mg/mL), 10 ((IC50 = 0.130 0.037 mg/mL)
and 15 (IC50 = 0.144 0.0002 mg/mL) show a considerable antiox-
Figure 1. Inhibition (%) of acethylcholinesterase activity by compounds 5–10 and
15–18.
Figure 4. Genotoxicity of compounds 7, 8, 10, 16, and 18.

50
J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
solution of the respective aldehyde (1 mmol) in anhydrous ethanol
3. Conclusions
(3 mL). The reaction mixture was heated at 75 °C for 5 h and then
72 h at rt. After solvent evaporation the residue was washed with
ethyl acetate and filtered off. Evaporation of the solvent afforded
a residue, which was purified by CC to give the corresponding 2-
oxo-/2-thioxopyrimidines.
Method C: A solution of urea/thiourea (1.43 mmol) in ethyl ace-
toacetate (2.14 mmol, 0.279 g, 0.3 mL) was added at rt, to a solu-
tion of the corresponding aldehyde (1 mmol) in anhydrous
ethanol (0.3 mL). The reaction mixture was submitted to micro-
wave, at 300 W for 10 min. Evaporation of the solvent afforded a
residue that was washed with ethyl acetate and filtered off. Evap-
oration of the solvent gave a residue, which was purified by CC to
give the corresponding 2-oxo-/2-thioxopyrimidines.
Synthetic approaches for new 4-glycosyl(oxo- and thi-
oxo)pyrimidines and 4-glycosyl-1H-tetrazoles starting from dialdo
sugars were exploited and standard thermal heating/flash heating
with microwave irradiation were used for the construction of the
heterocycle. The latter heating source proved to be the most appro-
priate leading to the target molecules in very high yield and in a
short time (10 min).
Most of the studied compounds inhibited acetylcholinesterase to
some extent (20–83% inhibition at 100 lg/mL compound concentra-
tion). The thioxopyrimidine derivatives seem to be more effective
than the oxopyrimidine analogues with the same glycosyl moiety,
and the most active ones have this heterocycle linked to furanose
moieties. They also showed antioxidant activity higher than that
of gallic acid for the b-carotene/linoleic acid assay. The tetrazole
linked to a 1,2-O-isopropylidene-xylosyl moiety unprotected at po-
sition 3 also showed considerable inhibition but low antioxidant
activity. With the exception of the cytotoxic 2-thioxopyrimidine
derivative 8, the bioactive compounds do not present genotoxicity
nor cytotoxicity. Hence, this family of compounds shows some
promise regarding their application as acetylcholinesterase inhibi-
tors for the control of neurodegenerative disease symptoms.
4.2.1. Ethyl (4R)-[(1R,2R,3S,4S)-3-O-benzyl-1,2-O-
isopropylidene-tetrofuranos-4-yl]-1,2,3,4-tetrahydro-6-methyl-
2-oxopyrimidine-5-carboxylate (5)
CC eluted with ethyl acetate/toluene 1/6 (v/v).Yield: (Method
A = 77%, 2.27 g; Method B = 80%, 2.35 g) Rf = 0.38 (ethyl acetate/tol-
uene 5/1); syrup; IR (neat) cmꢀ1: 3400; 3240 (N–H), 1700; 1680
(C@O), 1640 (C@C); ½a D25
ꢃ
¼ þ190ꢄ (c 0.5, CHCl3) U.V. (kmáx. nm) eth-
anol: 284 (e = 8935), 208 (e = 14413). Anal. Calcd for (C21H28O7N2):
C, 59.99; H, 6.71; N, 6.66. Found: C, 59.60; H, 6.84; N, 6.31. 1H NMR
(d ppm, J Hz, CDCl3): 8.92 (s, 1H, NH-1), 7.33–7.30 (m, 5H, Ph of
4. Experimental
4.1. Chemistry
Benzyl), 6.01 (d, 1H, H-10, J1 ,2 = 2.9), 5.66 (d, 1H, NH-3, J3,4 = 1.3),
0
0
4.76 (AB, 1H, CH2 of Benzyl, J = 12), 4.62 (d, 1H, H-20, J2 ,1 = 2.9),
4.57 (d, 1H, H-4, J4,3 = 1.3), 4.43 (AB, 1H, CH2 of Benzyl, J = 12),
0
0
4.28 (d, 1H, H-40, J4 ,3 = 4.4), 4.11 (q, 2H, CH2CH3, J = 7), 4.03 (d,
0
0
Melting points were determined with a melting point apparatus
(Leitz-Biomed) with platinum plate and are uncorrected. Optical
rotations were measured with an Atago Polax-D polarimeter and
IR spectra were recorded with a FT-IR Mattson Genesis II spectro-
photometer. 1H NMR spectra were run with a Bruker AC-250 P
spectrometer (250 MHz). Chemical shifts are expressed in parts
per million downfield from TMS. The 13C NMR spectra were run
at 62.90 MHz in the same spectrometer. The experiments were
performed in chloroform-d. Elemental analyses were obtained
with a Vario EL CHN analyser and were in good agreement
( 0.4%) with the calculated values. Domestic microwave oven
(Electrolux, 850 W, 20 L capacity) was used in the reactions carried
out under microwave irradiation. The progress of all reactions was
monitored by thin layer chromatography (TLC) using aluminum
sheets precoated with silica gel 60F254 to a thickness of 0.2 mm
(Merck). Preparative TLC was performed with aluminum plates
coated with silica gel 60F254 to a thickness of 0.5 mm (Merck).
Compounds were detected with UV light (254 nm) and/or by
spraying the sheets with a 3% vanillin in ethanol/sulfuric acid
(100 mL/1.5 mL) followed by heating. Column chromatography
(CC) was conducted under low pressure by elution of the columns
filled with silica gel (0.040–0.063 mm, Merck). All other chemicals
and solvents used were obtained from commercial sources and
used as received or dried using standard procedures.
1H, H-30, J3 ,4 = 4.4), 2.26 (s, 3H, 6-CH3), 1.43 and 1.30 (2s,
2 ꢂ 3H, 2 ꢂ CH3-isop), 1.18 (t, 3H, CH2CH3, J = 7). 13C NMR (d
ppm, CDCl3): 165.8 (Cq, C@O of CO2Et, 155.0 (Cq, C-2), 149.3 (Cq,
C-6), 137.1 (Cq-Ph of Benzyl), 128.6, 128.1, 127.7 (CH-Ph of Ben-
zyl), 111.6 (Cq, Cisop), 105.2 (CH, C-10), 97.0 (Cq, C-5), 84.3 (CH,
C-30), 83.4 (2 CH, C-20, C-40), 71.9 (CH2 of Benzyl), 59.6 (CH2CH3),
51.4 (CH, C-4), 26.8 and 26.2 (2 ꢂ CH3-Cisop), 18.4 (6-CH3), 14.2
(CH2CH3)).
0
0
4.2.2. Ethyl (4R)-[(1R,2R,3R,4S)-3-O-benzyl-1,2-O-
isopropylidene-tetrofuranos-4-yl]-1,2,3,4-tetrahydro-6-methyl-
2-oxopyrimidine-5-carboxylate (6)
CC eluted with ethyl acetate/toluene 1/5 (v/v). Yield: (Method
A = 79%, 2.32 g; Method B = 80%, 2.35 g); Melting point: 155.5–
157 °C; Rf = 0.64 (ethyl acetate/toluene: 8/1); ½a D25
ꢃ
¼ þ29ꢄ (c 3.25,
CHCl3); IR (KBr) cmꢀ1: 3320, 3220 (N–H), 1710; 1670 (C@O),
1630 (C@C); U.V. (kmáx nm) ethanol: 282
(e = 10304), 209
(e = 13365); Anal. Calcd for (C21H28O7N2): C, 59.99; H, 6.71; N,
6.66. Found: C, 59.89; H, 6.70; N, 6.68. 1H NMR (d ppm, J Hz,
CDCl3): 8.85 (s, 1H, NH-1), 7.36–7.23 (m, 5H, Ph Benzyl), 6.61 (s,
0
0
1H, NH-3), 5.70 (d, 1H, H-1, J1 ,2 = 3.2), 4.70 (AB, 1H, CH2 Benzyl,
J = 11.7), 4.56–4.46 (m, 3H, H-20, H-4, 1H-CH2 of Benzyl), 4.19–
4.06 (m, 3H, H-40 and CH2CH3), 3.83 (dd, 1H, H-3, J2 ,3 = 4.1 and
0
0
0
0
J3 ,4 = 8.6), 2.22 (s, 3H, 6-CH3), 1.53 and 1.29 (2s, 2 ꢂ 3H, 2 ꢂ CH3-
isop), 1.17 (t, 3H, CH2CH3, J = 7). 13C NMR (d ppm, CDCl3): 165.2
(Cq, C@O of CO2Et), 154.4 (Cq, C-2), 148.4 (Cq, C-6), 137.1 (Cq of
Benzyl), 127.4, 126.8, 126.6 (CH-Ph of Benzyl), 112.0 (Cq, Cisop),
103.2 (CH, C-10), 96.2 (Cq, C-5), 79.5 (CH, C-40), 77.4 (CH, C-30),
76.6 (CH, C-20), 70.9 (CH2 of Benzyl), 59.1 (CH2CH3), 51.2 (CH, C-
4), 25.8 and 25.7 (2 ꢂ CH3, Cisop), 18.0 (6-CH3), 13.4 (CH2CH3).
4.2. General procedure to obtain oxo-/thioxopyrimidines
Method A: A solution of urea/thiourea (1.43 mmol) in ethyl ace-
toacetate (2.14 mmol, 0.279 g, 0.3 mL) was added at rt to a solution
of the respective aldehyde (1 mmol) in anhydrous ethanol (3 mL).
The reaction mixture was heated at 75 °C for 5 h. Cooling to rt
and solvent evaporation afforded a residue that was washed with
ethyl acetate and filtered off. Evaporation of the solvent afforded
a residue, which was purified by CC to give the corresponding 2-
oxo-/2-thioxopyrimidines.
4.2.3. Ethyl (4R)-[(1R,2R,3S,4S,5S)-1,2:3,4-Di-O-isopropylidene-
pentopyranos-5-yl)-1,2,3,4-tetrahydro-6-methyl-2-
oxopyrimidine-5-carboxylate (7)
CC eluted with ethyl acetate/toluene 1/5 (v/v). Yield: (Method
B) = 82% (0.338 g); (Method C) = 84% (0.346 g); mp: 231–232° C;
Rf = 0.34 (ethyl acetate/toluene: 5/1); IR (net) (cmꢀ1): 3420
Method B: A solution of urea/thiourea (1.43 mmol) in ethyl
acetoacetate (2.14 mmol, 0.279 g, 0.3 mL) was added at rt, to a

J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
51
(N–H), 3254 (N–H), 1720 (C@O), 1712 (C@O ester), 1645 (C@C).
Anal. Calcd for (C19H23O8N2): C, 55.33; H, 6.84; N, 6.79; O, 31.03.
Found: C, 55.34; H, 6.85; N, 6.78; O, 31.02. 1H NMR (d ppm, J Hz,
CDCl3): 7.47 (s, 1H, NH-1), 5.82 (s, 1H, NH-3), 5.55 (d, 1H, H-10,
NMR (d ppm, J Hz, CDCl3): 7.80 (s, 1H, NH-1), 7.48 (s, 1H, NH-3),
5.57 (d, 1H, H-10, J1,2 = 5); 4.63 (d, 1H, H-4, J4,5 = 6.0), 4.57 (dd,
0
1H, H-50, J5 ,4 = 3.0), 4.34–4.28 (m, 2H, H-40 and H-20), 4.17–4.13
(m, 2H, CH2CH3), 3.70 (s, 1H, H-30), 2.36 (s, 3H, 6-CH3), 1.61, 1.36,
1.33, 1.30 (4 s, 4 ꢂ 3H, CH3 isop), 1.26 (t, 3H, CH2CH3, J = 7.0). 13C
NMR (d ppm, CDCl3): 174.6 (Cq, C-2), 165.7 (Cq, C@O of CO2Et),
145.8 (Cq, C-6), 110.0 and 108.5 (Cq, 2 isop), 98.3 (Cq, C-5), 96.4
(CH, C-10), 73.7 (CH, C-40), 71.6 (CH, C-20), 70.4 (CH, C-30), 68.7
(CH, C-50), 60.2 (CH2CH3). 54.0 (CH, C-4), 26.4, 25.8, 25.0, 24.3 (4
CH3, 2 isop), 18.3 (CH3), 14.3 (CH2CH3).
0
0
J1,2 = 5), 4.60 (d, 1H, H-4, J4,5 = 5.2 Hz), 4.57 (dd, 1H, H-50,
0
J5 ,4 = 2.5 Hz), 4.30–4.27 (m, 2H, H-40 and H-20), 4.20–4.09 (m, 2H,
CH2CH3), 3.73 (s, 1H, H-30), 2.34 (s, 3H, 6-CH3), 1.54, 1.48, 1.37,
1.33 (4 s, 4 ꢂ 3H, CH3 isop); 1.26 (t, 3H, CH2CH3, J = 7.0 Hz). 13C
NMR (d ppm, CDCl3): 174.9 (Cq, C@O of CO2Et), 165.2 (Cq, C-2),
149.4 (Cq, C-6), 110.1, 108.6 (Cq, 2 isop), 98.4 (Cq, C-5), 96.5 (CH,
C-10), 74.1 (CH, C-40), 72.3 (CH, C-20), 70.5 (CH, C-30), 68.8 (CH, C-
50), 60.2 (CH2CH3), 54.0 (CH, C-4), 26.3, 25.3, 25.0, 24.2 (4 CH3, 2
isop), 18.4 (6-CH3), 14.3 (CH2CH3).
0
0
4.3. General procedure to obtain nitriles
A solution of the appropriate aldehyde (7.25 mmol) in pyridine
(4 mL) was slowly added to a solution of hydroxylamine hydro-
chloride (7.93 mmol, 0.55 g) in water (2 mL). After stirring for
15 min at rt, pyridine (0.4 mL) was added to dissolve the precipi-
tated oxime. The mixture remained under stirring at rt for 1 h. Cu-
SO4ꢁ5H2O (14.4 mmol, 3.6 g) was added, followed by a solution of
triethylamine (14.4 mmol, 2 mL) in dichloromethane (3.6 mL)
and DCC (8.72 mmol, 1.8 g) in dichloromethane (15 mL). After 2 h
at rt, formic acid (1.3 mL) was added to eliminate DCC. The mixture
was filtered, and the filtrate was concentrated under vacuum. The
residue was treated with water (50 mL), and then extracted with
dichloromethane (3 ꢂ 50 mL). The organic phase was washed with
a solution of HCl 5% (v/v) (50 mL), dried with sodium sulfate, and
concentrated under vacuum to give the nitrile.
4.2.4. Ethyl (4R)-[(1R,2R,3S,4S)-3-O-benzyl-1,2-O-
isopropylidene-tetrofuranos-4-yl]-1,2,3,4-tetrahydro-6-methyl-
2-thioxo pyrimidine-5-carboxylate (8)
CC eluted with ethyl acetate/toluene 1/6 (v/v). Yield (Method
B) = 78% (0.349 g); (Method C) = 80% (0.358 g) mp: 135–136 °C;
Rf = 0.54 (ethyl acetate/toluene: 5/1); IR (net) (cmꢀ1): 3422, 3255
(N–H), 1721 (C@S), 1716 (C@O ester), 1642 (C@C). Anal. Calcd for
(C22H28O6N2S): C, 58.91; H, 6.29; N, 6.25; S, 7.15; O, 21.40. Found:
C, 58.92; H, 6.28; N, 6.24; S, 7.16; O, 21.41. 1H NMR (d ppm, J Hz,
CDCl3): 7.68 (s, 1H, NH-1); 7.42–7.22 (m, 5H, Ph of Benzyl), 7.18
(s, 1H, NH-3), 6.04 (d, 1H, H-10, J1,2 = 3.9 Hz). 4.84 (AB, 1H, CH2 of
Benzyl, J = 12.1), 4.66 (d, 1H, H-20, J = 3.9), 4.55–4.48 (m, 2H, H-4,
1H of CH2 of Benzyl), 4.29 (d, 1H, H-40, J3 ,4 = 5.1), 4.21–4.05 (m,
3H, CH2CH3 and H-30), 2.27 (s, 3H, 6-CH3), 1.46, 1.32, (2 s, 2 ꢂ 3H,
CH3 isop); 1.21 (t, 3H, CH2CH3, J = 7.6). 13C NMR (d ppm, CDCl3):
176.6 (Cq, C-2), 165.4 (Cq, C@O of CO2Et), 145.7 (Cq, C-6), 136.4
(Cq, Ph of Bn), 128.5, 128.2, 127.5 (3 ꢂ CH, Ph of Bn), 111.9 (Cq, 2
isop), 105.1 (CH, C-10), 98.5 (Cq, C-5), 83.8 (CH, C-30), 82.6 (CH, C-
20), 82.3 (CH, C-40), 71.8 (CH2, Benzyl), 60.0 (CH2CH3), 52.1 (CH,
C-4); 26.8; 26.2 (2 CH3, isop); 18.2 (6-CH3), 13.8 (CH2CH3).
0
0
4.3.1. 3-O-Benzyl-1,2-O-isopropylidene-a-D-
xylofuranosurononitrile (11)
After purification by CC eluted with ethyl acetate/toluene (1/1)
the nitrile was obtained in 75% (0.206 g) yield, as syrup, Rf = 0.77
(ethyl acetate/toluene 1/2). IR (net) (cmꢀ1): 2257 (CN) cmꢀ1
,
½
a 2D5
ꢃ
¼ þ13ꢄ (c 2.5, CHCl3). Anal. Calcd for (C15H17O4N): C, 65.44;
H, 6.22; N, 5.09; O, 23.25. Found: C, 65.43; H, 6.23; N, 5.10; O,
23.26. 1H NMR (d ppm, J Hz, CDCl3): 7.39–7.27 (m, 5H, Ph of Ben-
zyl), 5.98 (d, 1H, H-1, J1,2 = 3.3), 4.84 (d, 1H, H-4 J3,4 = 3.2 Hz),
4.73 (s, 2H, CH2 of Benzyl), 4.60 (d, 1H, H-3, J4,3 = 3.2), 4.16 (d,
1H, H-2, J2,1 = 3.3), 1.45, 1.30 (2 s, 2 ꢂ 3H, 2 ꢂ CH3 isop). 13C NMR
(d ppm, CDCl3): 136.6 (Cq, Ph of Benzyl), 128.7, 128.3, 128.1 (CH,
Ph of Benzyl), 114.1 (Cq, CN), 113.9 (Cq, isop), 105.7 (CH, C-1),
82.0 (CH, C-3), 81.6 (CH, C-2), 72.7 (CH2 of Benzyl), 69.5 (CH, C-
4), 27.0, 26.2 (2 ꢂ CH3 isop).
4.2.5. Ethyl (4R)-[(1R,2R,3R,4S)-3-O-benzyl-1,2-O-
isopropylidene-tetrofuranos-4-yl]-1,2,3,4-tetrahydro-6-methyl-
2-thioxo pyrimidine-5-carboxylate (9)
CC eluted with ethyl acetate/toluene 1/5 (v/v). Yield (Method
B) = 80% (0.358 g); (Method C) = 81% (0.363 g) m p: 109–110 C;
Rf = 0.49 (ethyl acetate/toluene: 5/1); IR (net) (cmꢀ1): 3419, 3251
(N–H), 1723 (C@S), 1714 (C@O of ester), 1639 (C@C). Anal. Calcd
for (C22H28O6N2S): C, 58.91; H, 6.29; N, 6.25; S, 7.15; O, 21.40.
Found: C, 58.90; H, 6.30; N, 6.26; S, 7.14; O, 21.41. 1H NMR (d
ppm, J Hz, CDCl3): 8.44 (s, 1H, NH-1), 7.46 (s, 1H, NH-3), 7.36–
7.14 (m, 5H, Ph of Benzyl), 5.68 (d, 1H, H-10, J1,2 = 3.2), 4.73 (AB,
1H, CH2 of Benzyl, J = 11.5), 4.57 (d, 1H, H-20), 4.55 (d, 1H, H-4,
4.3.2. 3-O-Benzyl-1,2-O-isopropylidene-a-D-ribofuranosurono-
nitrile (12)4
Yield = 78% (1.54 g), mp 123–124 °C; Rf = 0.74 (ethylacetate/tol-
0
J4,5 = 6.0), 4.50 (AB, 1H, CH2 of Benzyl, J = 11.5), 4.19–4.02 (m,
uene 1:2); ½a 2D5
ꢃ
¼ þ59 (c 1.9, CHCl3); IR (KBr) (cmꢀ1): 2117 (CN);
3H, CH2CH3 and H-40), 3.80 (dd, 1H, H-30, J2 ,3 = 4 Hz, J3 ,4 = 8.9),
2.25 (s, 3H, 6-CH3), 1.51, 1.29, (2 s, 2 ꢂ 3H, CH3 isop), 1.08 (t, 3H,
CH2CH3, J = 7.6). 13C NMR (d ppm, CDCl3): 176.3 (Cq, C-2), 165.4
(Cq, C@O of CO2Et), 145.8 (Cq, C-6), 137.6 (Cq, Ph of Benzyl)
129.0, 128.2, 127.9 (3 ꢂ CH, Ph of Benzyl), 113.1 (Cq, 2 isop),
104.1 (CH, C-10), 99.0 (Cq, C-5), 80.3 (CH, C-40), 77.3 (CH, C-30),
77.2 (CH, C-20), 71.9 (CH2, of Bn), 60,4 (CH2CH3), 52.0 (CH, C-4),
26.8, 26.5 (2 CH3, isop), 18.5 (6-CH3), 14.2 (CH2CH3).
UV (kmax nm) ethanol: 209 (e = 6887); Anal. Calcd for C15H17O4N:
0
0
0
0
C, 65.44; H, 6.22; N, 5.09. Found: C, 64.95; H, 6.38; N, 5.52. 1H
NMR (250 MHz): 7.32 (s, 5H, Ph), 5.74 (d, 1H, H-1, J1,2 = 3.4),
4.78, 4.71 (AB system, OCH2, Bn), 4.62 (d, 1H, H-4, J3,4 = 9), 4.53
(t, 1H, H-2, J2,3 = 4.1), 4.10 (dd, 1H, H-3), 1.56 (s, 3H, CH3 isop),
1.33 (s, CH3 isop); 13C NMR: 136.08 (Cq, Ph), 128.45, 128.23,
127.89 (CH, Ph), 116.82 (Cq, C-5), 113.98 (Cq, isop), 104.56 (CH,
C-1), 80.03 (CH, C-3), 76.74 (CH, C-2), 72.55 (OCH2, Bn), 66.07
(CH, C-4), 26.59 (CH3, isop), 26.05 (CH3, isop).
4.2.6. Ethyl (4R)-[(1R,2R,3S,4S,5S)-1,2:3,4-Di-O-isopropylidene-
pentopyranos-5-yl)-1,2,3,4-tetrahydro-6-methyl-2-thioxo
pyrimidine-5-carboxylate (10)
4.3.3. 1,2:3,4-Di-O-isopropylidene-a-D-galactopyranosurono-
nitrile (13)
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield (Method
B) = 79% (0.338 g); yield (Method C) = 81% (0.347 g); mp: 107–
108° C; Rf = 0,31 (ethyl acetate/toluene: 5/1); IR (net) (cmꢀ1):
3420, 3254 (N–H), 1718 (C@S), 1714 (C@O ester), 1640 (C@C).
Anal. Calcd for (C19H28O7N2S): C, 55.26; H, 6.59; N, 6.54; S, 7.48;
O, 26.15. Found: C, 55.25; H, 6.58; N, 6.53; S, 7.49; O, 26.15; 1H
CC eluted with ethyl acetate/toluene 1/4 (v/v).Yield 78%
(0.199 g), syrup, Rf = 0.55 (ethyl acetate/toluene 1/2); IR (neat)
(cmꢀ1): 2259 (CN). Anal. Calcd for (C12H17O5N): C, 56.46; H, 6.71;
N, 5.49; O, 31.34. Found: C, 56.45; H, 6.70; N, 5.50; O, 31.35. 1H
NMR (d ppm, J Hz, CDCl3): 5.53 (d, 1H, H-1, J1,2 = 4.9), 4.69–4.65
(m, 2H, H-3 and H-5), 4.39–4.32 (m, 2H, H-2 and H-4), 1.54, 1.53,

52
J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
1.38, 1.34 (4s, 4 ꢂ 3H, 4 ꢂ CH3 isop). 13C NMR (d ppm, CDCl3):
115.1 (Cq, C-6), 110.8 and 109.2 (2 ꢂ Cq, isop), 95.8 (CH, C-1),
70.4 (CH, C-3), 70.0 (CH, C-2), 69.5 (CH, C-4), 59.8 (CH, C-5), 25.6,
25.6, 24.3 and 24.2 (4 ꢂ CH3 isop).
(297 mg), solid; mp 235–236 °C Rf = 0.80 (ethyl acetate/methanol
1/2), IR (KBr) 3479 (C@N) cmꢀ1, ½a D17
¼ ꢀ5:92 (c 19.5, CH3COCH3).
ꢃ
FAB(+)-MS m/z: 299 (M+1)+ (12), 283 (MꢀCH3)+ (100). 1H NMR [(d
ppm, J Hz, (CD3)2CO): 5.54 (d, 1H, H-10, J1,2 = 4.9), 5.25 (d, 1H, H-40,
J3 ,4 = 1.96), 4.70 (dd, 1H, H H-50, J = 5.2 and 7.7), 4.49 (dd, 1H, H-30,
J = 2.4 and 7.8), 4.40 (dd, 1H, H-20, J = 2.4 and 4.9), 1.54, 1.53 1.38
and 1.34 (4s, 4 ꢂ 3H, 4 ꢂ CH3 isop). 13C NMR [d ppm, (CD3)2CO):
154.5 (Cq, C-5), 110.3, 109.8 (2 ꢂ Cq, isop), 97.2 (CH, C-10), 72.9
(CH, C-30), 71.3 (CH, C-20), 71.2 (CH, C-40), 64.6 (CH, C-50), 26.3,
26.0, 24.9 and 24.2 (4 ꢂ CH3 isop).
0
0
4.3.4. 1,2-O-Isopropylidene-a-D-xylofuranosurononitrile (14)
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield 75%
(0.139 g), syrup, Rf = 0.44 (ethyl acetate/toluene 1/2); IR (neat)
(cmꢀ1): 2259 (CN). Anal. Calcd for (C8H11ON): C, 51.89; H, 5.99;
N, 7.56; O, 34.56. Found: C, 51.88; H, 5.98; N, 7.57; O, 34.57. 1H
NMR (d ppm, J Hz, CDCl3): 6.01 (d, 1H, H-1, J1,2 = 3.4), 4.99 (d, 1H,
H-4 J4,3 = 2.7), 4.59 (d, 1H, H-2, J2,1 = 3.4), 4.47 (d, 1H, H-3,
J3,4 = 2.7), 3.76 (br s, 1H, OH-3), 1.47, 1.31 (2s, 2 ꢂ 3H, 2 ꢂ CH3
isop). 13C NMR (d ppm, CDCl3): 115.3 (Cq, CN), 112.8 (Cq, isop),
105.2 (CH, C-1), 83.7 (CH, C-3), 74.9 (CH, C-2), 70.7 (CH, C-4),
26.5 and 25.7 (2 ꢂ CH3 isop).
4.4.4. 5-[(1R,2R,3S,4S)-1,2-O-isopropylidenetetrofuranos-4-
yl]tetrazole (18)
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield (Method
1) = 98% (225 mg), (Method 2) = 99% (228 mg), (Method 3) = 99%
(228 mg); solid, mp 154–155 °C Rf = 0.60 (ethyl acetate/methanol
1/2), IR (KBr) cmꢀ1 3583 (OH), 3477 (NH), ½a 1D7
ꢃ
¼ ꢀ3:15ꢄ (c 10,
4.4. General procedure for preparation of tetrazoles
CH3COCH3). ESI(+)-HRMS m/z: 251.0755 (M+Na)+ (61) (251.0757
calc for C15H18N4O4Na); 229.0936 (M+H)+ (100) (229.0938 calc.
for C15H19N4O4). 1H NMR [d ppm, J Hz, (CD3)2CO]: 6.08 (d, 1H, H-
10, J1,2 = 3.2 Hz), 5.62 (d, 1H, H-40, J3,4 = 2.6), 4.72 (d, 1H, H-30,
J4,3 = 3.3), 4.49 (d, 1H, H-20, J2,1 = 3.2), 1.50 and 1.32 (2s, 2 ꢂ 3H,
2 ꢂ CH3 isop). 13C NMR [d ppm, (CD3)2CO]: 153.6 (Cq, C-5), 112.8
(Cq, isop), 106.0 (CH, C-10), 85.9 (CH, C-30), 76.2 (CH, C-20), 75.8
(CH, C-40), 27.0 and 26.3 (2 ꢂ CH3 isop).
Method 1: A mixture of the nitrile (1.0 mmol) in anhydrous DMF
(2 mL), sodium azide (1.1 mmol, 72 mg) and ammonium chloride
(1.3 mmol, 70 mg) was refluxed (120 °C) under stirring for 3 h (un-
til TLC showed complete conversion). After solvent evaporation,
the residue was submitted to flash chromatography eluted with
ethyl acetate/toluene 1/2 (v/v).
Method 2: A mixture of the nitrile (1 mmol) in water (3.0 mL),
sodium azide (1.05 mmol, 68 mg) and zinc chloride (1.1 mmol,
15 mg) was refluxed (100 °C), under stirring for 2.5 h (until TLC
showed complete conversion). After solvent evaporation, the resi-
due was submitted to flash chromatography eluted with ethyl ace-
tate/toluene 1/2 (v/v).
Method 3: A mixture of the nitrile (1 mmol) in water (3 mL), so-
dium azide (1.05 mmol. 68 mg), and zinc chloride (1.1 mmol,
15 mg) was heated under microwave irradiation (100 W) for
10 min (until TLC showed complete conversion). After solvent
evaporation, the residue was submitted to flash chromatography
eluted with ethyl acetate/toluene 1/2 (v/v).
4.5. Biological activity
4.5.1. Enzymatic studies
A double beam spectrophotometer ShimadzuÒ equipped with
thermostatic cell holders was used on visible range and operated
on the kinetic mode for the enzymatic studies. The absorbance data
were acquired in a computer by means of UV Probe software.
Appropriate disposable plastic cuvettes PlastibrandÒ were used
in the kinetic experiments. The following materials were pur-
chased from Aldrich: enzyme acetylcholinesterase (AChE) from bo-
vine erythrocytes, substrate acetylthiocholine iodide (ATChI), and
5,50-dithiobis-2-nitrobenzoic acid (DTNB). Other Aldrich reagents
used for the preparation of buffers and solutions were KH2PO4,
KOH and NaHCO3. Deionized water was used to prepare the buffer
pH 8.0, the substrate and DTNB solutions.
4.4.1. 5-[(1R,2R,3S,4S)-3-O-benzyl-1,2-O-isopropylidene-
tetrofuranos-4-yl]tetrazole (15)
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield (Method
1) = 96% (305 mg), (Method 2) = 98% (312 mg), (Method 3) = 98%
(312 mg), solid, mp 139–140 °C; Rf = 0.81 (ethyl acetate/methanol
4.5.1.1. Solutions preparation.
Preparation of 0.1 M phos-
1/1), IR (KBr) cmꢀ1 3482 (NH), ½a D17
ꢃ
¼ þ1:58ꢄ (c 1.8, CH3COCH3).
phate buffer pH 8.0: KH2PO4 (136.1 mg) was dissolved in water
(10 mL) and adjusted with KOH to a pH of 8.0 0.1. Buffer was
freshly prepared and stored below 5 °C.
AChE solution 1.32 U/mL: the enzyme (1.02041 U, 4.4 mg) was
dissolved in freshly prepared buffer pH 8.0 (1.0 mL).
DTNB solution 0.01 M: DTNB (3.96 mg) was dissolved in water
(1 mL) containing sodium hydrogen carbonate (1.5 mg).
ATChI solution 0.022 M: ATChI (6.4 mg) was dissolved in water
(1 mL).
All solutions were stored in Eppendorf caps (100 lL aliquots) in
the refrigerator at below 5 °C. The pure compounds were dissolved
in DMSO and diluted with distilled water until 4.4 mg/mL concen-
tration to obtain the final compound concentration of 100 lg/mL in
ESI(+)-HRMS: m/z 341.1228 (M+Na)+ (63), (341.1226 calcd for
C
C
15H18N4O4Na) 319.1406 (M+H)+ (100) (319.1407 calc for
15H19N4O4). 1H NMR (d ppm, J Hz, CDCl3): 7.29–7.26 (m, 3H, Ph
of Benzyl), 6.99–6.96 (m, 2H, Ph of Benzyl), 6.08 (d, 1H, H-10,
J1 ,2 = 3.6), 5.74 (d, 1H, H-40, J3 ,4 = 3.16), 4.74 (d, 1H, H-20,
0
0
0
0
J2 ,1 = 3.6), 4.47 (AB, CH2 of Benzyl, J = 11.3 Hz), 4.30 (d, 1H, H-30
0
0
0
0
J2 ,3 = 3.07 Hz), 4.26 (AB, CH2 of Benzyl, J = 11.3 Hz), 1.55 and 1.37
(2s, 2 ꢂ 3H, 2 ꢂ CH3 isop). 13C NMR (d ppm, CDCl3): 152.6 (Cq, C-
5), 136.1 (Cq, Ph of Benzyl), 128.8, 128.5 and 127.9 (CH, Ph of Ben-
zyl), 113.2 (Cq, isop), 105.5 (CH, C-10), 82.6 (CH, C-30 and C-20), 74.6
(CH, C-40), 72.9 (CH2 of Benzyl), 27.0, 26.3 (2 ꢂ CH3 isop).
4.4.2. 5-[(1R,2R,3R,4S)-3-O-Benzyl-1,2-O-
isopropylidenetetrofuranos-4-yl]tetrazole (16)4
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield (Method
1) = 97% (308 mg), (Method 2) = 99% (315 mg), (Method 3) = 98%
(312 mg).
the enzymatic test. No inhibition was detected by residual DMSO
(<0.5%) at the reaction cuvette.
4.5.1.2. AChE inhibitory activity assay.
fer (200 L), enzyme (5 L), DTNB (5
pound (5
in a heated water bath, and then the substrate ATChI solution
(5 L) was added to start the enzymatic reaction. The absorbance
A mixture of the buf-
l
l
lL) and new synthetic com-
lL) solutions was prepared and kept for 15 min at 30 °C
4.4.3. 5[(1R,2R,3S,4S,5S)-1,2:3,4-Di-O-isopropylidene-
pentopyranos-5-yl]tetrazole (17)
l
CC eluted with ethyl acetate/toluene 1/4 (v/v). Yield (Method
1) = 98% (294 mg), (Method 2) = 99% (297 mg), (Method 3) = 99%
was read along reaction time for 4 min under a controlled temper-
ature of 30 °C. At least four replicates were made. Several blank

J. A. Figueiredo et al. / Carbohydrate Research 347 (2012) 47–54
53
assays without the new synthetic compounds were carried out in
order to determine the average Vmax. Also assays without the en-
zyme and the inhibitor compound were carried out to check for
any non-enzymatic hydrolysis of the substrate. The final concen-
trations in the test were as follows: [AChE] = 0.03 U/mL, [com-
thin water film. Scoring was performed in a Zeiss optical micro-
scope at 1,250ꢂ magnification, by observing 100 complete meta-
phases (presenting 46 centromeres) per case. Classification of
chromosomal aberrations was done according to criteria described
in Rueff et al.47 The mitotic index was also quantified by counting
the number of metaphases per 1000 nuclei.
pound] = 100 lg/mL, [DTNB] = 0.0002273 M, [ATChI] = 0.0005 M.
References
4.5.2. b-Carotene/linoleic acid bleaching test
The antioxidant activity of pure compounds was evaluated
using a b-carotene-linoleic acid model system43 with a slight mod-
ification of the described procedure.44 Briefly, a solution of b-caro-
tene dissolved in chloroform (0.5 mL, 2 mg/mL) was pipetted into a
round-bottomed flask (50 mL) and Tween 40 (200 mg) was added.
After removing the chloroform with a rotary evaporator, aerated
1. McDonald, R. J.; Craig, L. A.; Hong, N. S. Behav. Brain Res. 2010, 214, 3–11.
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