Bioactive pseudo-C-nucleosides containing thiazole, thiazolidinone, and tetrazole rings

Article abstract of DOI:10.1081/CAR-200060396

The synthesis of new pseudo-C-nucleosides was accomplished starting from 3-O-benzyl-1,2-O-isopropylidene-α-D-ribo-pentodialdo-1,4-furanose, aiming to build thiazole, triazole, tetrazole, and thiazolidinone derivatives. The stereochemistry of the thiazolid

Full text of DOI:10.1081/CAR-200060396

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Bioactive Pseudo‐C‐nucleosides
Containing Thiazole, Thiazolidinone, and
Tetrazole Rings
Amélia P. Rauter a , Mária Padilha a , José A. Figueiredo b , Maria I.
Ismael b , Jorge Justino c , Humberto Ferreira d , Maria J. Ferreira d ,
Candasamy Rajendran e , Richard Wilkins e , Pedro D. Vaz a f & M. J.
Calhorda a f
a Departamento de Química e Bioquímica, Faculdade de Ciências da
Universidade de Lisboa, Campo Grande, Lisboa, Portugal
b Departamento de Química da Universidade da Beira Interior,
Unidade I&D de Materiais Têxteis e Papeleiros, Covilhã, Portugal
c Escola Superior Agrária—Instituto Politécnico de Santarém,
Santarém, Portugal
d Centro de Química Estrutural do Instituto Superior Técnico, Lisboa,
Portugal
e School of Biology, University of Newcastle, Newcastle upon Tyne,
UK
f ITQB, UNL, Oeiras, Portugal
Published online: 16 Aug 2006.
To cite this article: Amélia P. Rauter , Mária Padilha , José A. Figueiredo , Maria I. Ismael , Jorge
Justino , Humberto Ferreira , Maria J. Ferreira , Candasamy Rajendran , Richard Wilkins , Pedro D.
Vaz & M. J. Calhorda (2005): Bioactive Pseudo‐C‐nucleosides Containing Thiazole, Thiazolidinone, and
Tetrazole Rings, Journal of Carbohydrate Chemistry, 24:3, 275-296
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Journal of Carbohydrate Chemistry, 24:275–296, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200060396
Bioactive Pseudo-C-
nucleosides Containing
Thiazole, Thiazolidinone,
and Tetrazole Rings
´
´
Amelia P. Rauter and Maria Padilha
ˆ
´
´
Departamento de Quımica e Bioquımica, Faculdade de Ciencias da Universidade de
Lisboa, Campo Grande, Lisboa, Portugal
´
Jose A. Figueiredo and Maria I. Ismael
´
Departamento de Quımica da Universidade da Beira Interior, Unidade I&D de
ˆ
Materiais Texteis e Papeleiros, Covilha˜, Portugal
Jorge Justino
´
´
´
´
Escola Superior Agraria—Instituto Politecnico de Santarem, Santarem, Portugal
Humberto Ferreira and Maria J. Ferreira
´
´
Centro de Quımica Estrutural do Instituto Superior Tecnico, Lisboa, Portugal
Candasamy Rajendran and Richard Wilkins
School of Biology, University of Newcastle, Newcastle upon Tyne, UK
Pedro D. Vaz and M. J. Calhorda
ˆ
´
´
Departamento de Quımica e Bioquımica, Faculdade de Ciencias da Universidade de
Lisboa, Campo Grande, Lisboa, Portugal and ITQB, UNL, Oeiras, Portugal
Received September 9, 2004; accepted March 16, 2005.
Dedicated to the memory of Prof. Jacques van Boom.
´
´
´
Address correspondence to Amelia P. Rauter, Departamento de Quımica e Bioquımica,
ˆ
´
Faculdade de Ciencias da Universidade de Lisboa, Edifıcio C8, 58 Piso, Campo
Grande, 1749-016 Lisboa, Portugal. Tel.: þ 351 217500952; Fax: þ351 217500088;
E-mail: eaprauter@fc.ul.pt
275

A. P. Rauter et al.
276
The synthesis of new pseudo-C-nucleosides was accomplished starting from 3-O-benzyl-
1,2-O-isopropylidene-a-D-ribo-pentodialdo-1,4-furanose, aiming to build thiazole, triazole,
tetrazole, and thiazolidinone derivatives. The stereochemistry of the thiazolidinone
epimers was confirmed by DFT/B3LYP calculations. The well-known diversity of bio-
logical activities exhibited by compounds with these heterocyclic moieties in their struc-
ture encouraged the investigation of the insecticidal activity against Musca domestica.
The thiazole derivative was efficient as insecticide with LD50 ¼ 6.7 ng/g, which is in the
order of the LD50 values for pyrethroids on the same insect. All the compounds tested
showed knockdown, mediated through action on the insect nervous system. The neuro-
activity found in insects has motivated the evaluation of the inhibition of acetyl-
cholinesterase and butyrylcholinesterase. The two thiazolidinone derivatives tested
inhibited butyrylcholinesterase from human serum (36% and 22%), while inhibition of
amphiphilic acetylcholinesterase from human erythrocytes at the same concentrations
was found to be very low (5% and 8%). This selective activity may indicate potential
application of these structures for amelioration of Alzheimer’s disease.
Keywords Pseudo-C-nucleosides, Synthesis, DFT calculations, Insecticidal activity,
Neuroactivity, Alzheimer disease
INTRODUCTION
The use of C-nucleosides is well known in medicine, and the research into this
group of compounds is likely to yield new applications for essential needs in
medicine and other fields, such as pest management. This potential may be
further increased by investigating pseudo-C-nucleosides, the chemical struc-
ture of which differ from that of C-nucleosides in the position of the sugar to
which the heterocyclic moiety is bound. A variety of biological activities is
reported in the literature for these compounds. Tiazofurin, a 2-substituted
thiazole-4-carboxamide derivative, and a synthetic C-nucleoside, which has
demonstrated antiviral activity and potent antitumor activity against several
murine tumors, was submitted to clinical trials against lung cancer.[1–3] It
was also found to be a potent inducer of leukemia cell differentiation along
with other nucleotides containing the thiazole-4-carboxamide unit in their
structure.[4]
The thiazole unit is also a component of the structure of the macrocyclic
antibiotics thiocilline I[5] and leinamycin, an antitumor natural antibiotic,
which is known to promote DNA damage.[6] Other antitumor indolinones
contain an imidazothiazole system in their structure.[7]
2,4-Disubstituted thiazole-containing secondary metabolites were isolated
from marine sources,[8] including the cytotoxins patellazoles A–C[9] and patea-
mine,[10] the antimitotic agent curacin A,[11] and the potent anthelmintic agent
micothiazole.[12] An anthelmintic thiazole derivative is used to control
gastrointestinal nematodes in ruminants.[13]
4-Thiazolidinone derivatives are fungicidal,[14] antibacterial,[15] anti-
inflammatory,[15] analgesic,[15] antioxidant,[16] cytotoxic,[17] antiviral[18] and

Bioactive Pseudo-C-nucleosides
277
have potent antituberculosis activity against Mycobacterium tuberculosis H
(37) Rv (ATCC 27294).[14] These types of compounds are also known as
herbicides and have insecticidal activity against Anopheles sp.,[19] Culex
sp.,[19] and cockroaches.[20]
Ribavirin is a broad-spectrum antiviral agent, displaying antitumor
activity in mice.[21] Synthesis of this triazole nucleoside and its C-nucleoside
analogues[22] have attracted considerable attention due to their bioactivities.
Preparation of 1,2,4-1H-triazolated nucleosides was reported by site-specific
triazolation at C-4 of 5-substituted uridines with triazolide, formed from
1,2,4-triazole, POCl3, and triethylamine.[23] Triazole nucleosides were also
synthesised by dehydrative cyclization of pentitol-1-yl triazoles.[24] Triazolyl
moieties were found to be good leaving groups for the solid-phase synthesis
of oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases,
devised to be used in antigene strategies for the control of gene expression.[25]
Triazoles with leaf bleaching activity and germination promoting activity were
also described in the literature.[26]
The antifungal[27,28] and pesticidal[29] activities of tetrazoles have
encouraged the search for new compounds containing these units in their
structure. Tetrazole derivatives have also been used as promising activating
catalysts of phosphoramidites, resulting in rapid condensation with carbo-
xamides to give N-acylphosphoramidate linkages.[30] This linkage is common
in nucleotide antibiotics, namely, in phosmidosine, which is a specific inhibitor
of Botrytis cinerea.
As part of our ongoing search for new compounds derived from sugars
focusing on their potential pesticidal applications, we report now the synthesis
of new pseudo-C-nucleosides possessing thiazole, triazole, tetrazole, and thiazo-
lidinone moieties. In a preliminary evaluation of biological effects, compounds
3, 4, and 6 were tested in two invertebrate species, the crustacean Artemia
salina and the dipteran (insect) Musca domestica. This enabled limited com-
parison of juvenile aquatic and adult terrestrial arthropods based on whole
organism toxicity assays. The neuroactivity shown led us to accomplish prelimi-
nary experiments on their inhibition of amphiphilic acetylcholinesterase from
human erythrocytes and of butyrylcholinesterase from human serum,
enzymes that are involved in neurotransmission in the brain, and thus, this
type of compound may find value ameliorating Alzheimer’s disease.[31]
RESULTS AND DISCUSSION
Synthesis of Compounds 2–9
3-O-Benzyl-1,2-O-isopropylidene-a-D-ribo-pentodialdo-1,4-furanose (1) was
used as synthon for the preparation of the new pseudo-C-nucleosides described
in this work. The transformation of the aldehyde functionality into nitrile has

A. P. Rauter et al.
278
been reported in the literature, and the various methods available for that con-
version include oxidative transformation of the N,N-dimethylhydrazones,[32]
oxidation of aldimines,[33] dehydration of aldoximes by using agents such as
N-methylpyrrolidone,[34] solid-state conversion by alumina-supported potas-
sium peroxymonosulfate,[35] and treatment with hydroxylamine hydrochloride
under microwave irradiation,[36] among others. We report now the one-pot syn-
thesis of the sugar nitrile 2 in 78% yield, by reaction of 1 with hydroxylamine
hydrochloride and addition of triethylamine, copper(II) sulfate pentahydrate,
and the dehydration agent DCC[37] (Sch. 1). The IR band at 2117 cm21
together with the 13C NMR signal at d 116.82 confirmed the formation of the
sugar nitrile 2.
Reaction of 2 with H2S in chloroform in the presence of DMAP at 308C, led
to the thioamide derivative 3 in 85% yield. The IR spectrum presented the NH2
bands at 3447 and 3325 cm21. The singlets at d 7.77 and d 7.59 corresponding to
Scheme 1: Synthesis of thiazole, thiazolidinone, tetrazole, and triazole derivatives. Reagents
and conditions: (a) NH2OH.HCl, Pyr., H2O; CuSO4.5H2O, Et3N, DCC, CH2Cl2 (78%); (b) H2S,
DMAP, CHCl3 (85%); (c) BrCH2COCOOEt, CH3CN (50% for 4); NH3, MeOH (88% for 5); (d) NaN3,
DMF, NH3.HCl (97%); (e) PhCONH.NH2 (87.5%); (f) PhNH2, toluene, p-TsOH, powdered
˚
molecular sieve 4 A; (g) HSCH2COOH, toluene (57% for 8, 13% for 9, overall yield from 1).

Bioactive Pseudo-C-nucleosides
279
1
NH2 and the C55S signal at d 203.71 detected in the H NMR and 13C NMR
spectra, respectively, are in agreement with the introduction of the thioamide
moiety in the molecule.
The synthesis of a variety of thiazole-4-carboxylate derivatives was
described in the literature using ethyl isocyanoacetate and thiono esters[38] or
cyclodehydration of amides.[39] We have constructed the thiazole-4-carboxylate
unit using a classical method, by treatment of 3 with ethyl bromopyruvate in
acetonitrile at 08C[40,41] to give the corresponding thiazole-4-carboxylate 4 in
50% yield as major product, separated by flash chromatography from a
complex mixture of secondary products. Formation of the heterocyclic ring
was detected by the singlet at d 8.17 corresponding to H-5 and by the
13C NMR signals due to the olefinic carbon atoms C-2, C-4, and C-5 at
d 161.43, d 147.50, and d 128.29, respectively. The presence of the ethoxycarbo-
nyl group was confirmed by the resonances at d 4.20 for the methylene group
and the methyl group in a multiplet at d 1.36–1.43. The signals of the corre-
sponding carbon atoms appeared at d 61.47 and d 14.34, respectively, while
that of the carbonyl group was present at d 169.08.
Treatment of 4 with methanolic ammonia led to 5 in 88% yield. The
presence of the NH2 group was confirmed by its IR bands at 3480 and
3405 cm21 and by the singlet at d 5.82. The carbonyl group appeared at d
168.39 in the 13C NMR spectrum.
The synthesis of the tiazofurin analogue 5 was thus accomplished in three
steps with 37% overall yield, starting from the pentofuranose–4–nitrile 2, a
higher yield than that obtained previously for similar transformations,
leading to the synthesis of tiazofurin.[1]
The tetrazole derivative 6 was easily obtained by reaction of the nitrile 2
with sodium azide at 1008C in 97% yield. The assignment of H-40 at d 5.42
shifted downfield to the corresponding proton of the nitrile precursor, and the
presence of the C ¼ N signal at d 155.50 confirmed the structure proposed for 6.
Reaction of benzoyl hydrazide with the thioamide 3 gave the phenyl
1,2,4-triazole derivative 7 in 87.5% yield, following the procedure described
by Pesson et al.[42] The triazole moiety was characterized by the presence of
the signals at d 165.54 corresponding to C-5 and at d 163.16 corresponding to
C-3. The assignment was established by observation of the COLOC
spectrum, which presented a correlation between C-5 and the double doublet
at d 7.99, the resonance due to the protons at positions 2 and 6 of the phenyl
group bound to C-5, and the correlations of C-3 with H-40 at d 5.29 and with
H-30 at d 4.31. The presence of NH was confirmed by its IR band at 3490 cm21
.
Compound 1 reacted with aniline in the presence of p-toluenesulphonic
˚
acid and powdered molecular sieves 4 A to afford an intermediate phenylimine,
which was treated with sulfanylacetic acid in toluene to give a mixture of the
thiazolidin-4-one diastereoisomers 8 and 9, which could be separated by CC
1
and isolated in 57% and 13% yield, respectively. The H NMR experimental

A. P. Rauter et al.
280
Table 1: Comparison of experimental and calculated NMR chemical shifts using
the GIAO algorithm[50] and referenced to TMS, for diasteroisomers 8 and 9.
8
9
Proton
Exp.
Calc.
Exp.
Calc.
H-2
H-5a
5.37
5.14
3.19
3.76
5.82
4.52
3.68
4.00
4.51
5.12
4.85
4.40
3.32
3.76
4.99
4.33
3.65
4.34
4.54
4.92
3.42, 3.48
3.31, 3.36
5.71
4.56
3.91
4.28–4.36
4.28–4.36
4.61–4.65
7.31–7.38
3.38, 3.43
3.72–3.81
5.73
4.56
3.72–3.81
4.18
4.43, 4.39
4.72, 4.68
7.24–7.42
H-5b
H-10
H-20
H-30
H-40
CH2 (Bn)
CH2 (Bn)
Ph (Bn)
Ph (N)
7.18–7.52
7.43–7.82
7.38–7.83
7.13–7.74
data of both isomers is given in Table 1. The resonances of H-2 and H-5b, which
are on opposite sides of the heterocyclic ring (Fig. 1) are significantly different
in both isomers. The signal of H-5b is part B of an AB system, showing a
coupling constant with H-2 of 1.5 Hz in both compounds and appearing at d
3.31, 3.36 (for 8), and in the multiplet at d 3.72–3.81, together with the
signal of H-30 (for 9), while the signals of H-5a (part A of the AB system) do
not change substantially in both compounds. The configuration of the stereo-
genic center at C-2 in 8 was first inferred by means of NOE experiments. Detec-
tion of the interaction between H-5b and H-30 indicated that H-5b has the
orientation depicted in Figure 1. H-5a presented an interaction with H-2 at d
5.37 and did not show interaction with sugar protons, inferring that H-2 and
Figure 1: Some NOE interactions relevant for the structure elucidation of compounds 8 and 9.

Bioactive Pseudo-C-nucleosides
281
H-5a are on the same side of the thiazolidinone ring. These findings are in
agreement with the S configuration for C-2. NOE experiments for 9 showed
an interaction of H-2 at d 4.85 with the H-30 signal included in the multiplet
at d 3.73–3.78, which contains also H-5b. In addition a small interaction of
H2 with H-5a was observed, as expected.
To confirm the structure and NMR signals’ assignment for the thiazolidin-4-
one diastereoisomers 8 and 9, DFT (B3LYP/6-311Gꢀꢀ) calculations were per-
formed (see Computational Details). The methyl groups of the isopropylidene
group were substituted by hydrogen atoms, to simplify the models, since
they are not involved in the relevant interactions. The geometries of 8 and 9
were fully optimized and the minimum energy structures are presented
in Figure 2. Diastereoisomer 8 was found to be more stable than 9 by
16.2 kJ mol21, a difference low enough to predict both isomers to occur. NMR
chemical shifts were calculated using the GIAO option and the results show a
general good agreement with the experimental ones. A comprehensive list of cal-
culated and experimental values is given in Table 1. However, for compound 8
the experimental signal at d 3.31, 3.36, assigned to proton H-5b, is not consistent
with the calculated value at d 3.76. The experimental chemical shift of H-5b is
lower than that of H-5a, while for the calculated chemical shifts the opposite
was observed. This can be explained by the existence of an intramolecular
˚
C–H ꢁ ꢁ ꢁ O hydrogen bond (H ꢁ ꢁ ꢁ O distance ¼ 2.9 A) between the oxygen atom
of the furanose ring and this endocyclic proton attached to C-5 (see Fig. 2).
Such weak interactions are known to be underestimated by hybrid DFT
methods.[43] Therefore, the calculations confirm the assignment of the exper-
imental NMR data, showing also that the predicted configuration of the C-2
stereogenic center of isomers 8 and 9 is correct.
Biological Results
Toxicological assays against Musca domestica and Artemia salina
The aquatic bioassays using the brine shrimp larvae showed no effects at
all, even when the concentration of compounds 3, 4, and 6 was increased to
100 mg/L. This indicated a very low level of activity even if the uptake by
the shrimp was limited. This organism is often used for assessing environ-
mental toxicity and cytotoxicity and can be viewed as a reference species.
The results obtained with the housefly are summarized in Table 2. All three
compounds exhibited knockdown at the concentrations used. However,
compound 4 gave this effect at much lower dosages (ꢂ1023) and was the only
compound to be insecticidal. By performing regression analysis on the 24-hr
mortality data for this compound based on percent mortality and log dosage
(presented in Fig. 3) an estimate for the median lethal dosage is as
follows: LD50 ¼ 6.7 ng/g (0.67 mg/g body weight), 95% confidence limits

A. P. Rauter et al.
282
Figure 2: DFT energy minimized geometries of diastereoisomers 8 and 9 at the B3LYP/6-311Gꢀꢀ
level.

Table 2: Responses of adult houseflies to topical application of compounds 3, 4, and 6.
Time to
Dosage
(ng/
insect)
100%
knockdown
(min)
Duration of
knockdown
(min)
Time to 100%
recovery
(min)
% Mortality at
24 hr
Compound nr.
3 (Thioamide)
1
10
100
15–17
15–20
15–20
20–21
20–22
20–25
15–20
20–28
30–33
0
0
0
4 (Thiazole)
1
2
4
6
8
10
12
14
10–12
10–13
10–12
10–15
10–15
10–15
10–20
10–20
13–17
14–15
15–20
18–21
17–30
—
10–11
10–15
12–15
20–22
0
0
20
40
60
80
100
100
20–23
20% recovery
no recovery
no recovery
—
—
6 (Tetrazole)
1
10
100
15–20
15–20
15–19
8–10
10–12
15–20
5–10
10–12
15–20
0
0
0

A. P. Rauter et al.
284
Figure 3: Mortality response (probit percent) of adult housefly with thiazole solutions in
acetone applied topically.
4.8–9.3 ng/insect, which is in the order of the typical value for the LD50 value
for pyrethroids on the same insect (e.g., 0.3 ng/g for the highly potent deltame-
thrin).[44] For this compound complete knockdown was achieved at the lowest
dosage used (1.0ng/insect), and therefore the estimate for the median effective
dosage (ED50) for knockdown must be less than this, and there is likely to be a
factor of about 10 between the dosages for these two responses.
The other two compounds (3 and 6) were not toxic to the adult housefly,
causing no mortality at all up to 10 ng/insect (1 mg/g body weight). This
might be due to lack of cuticle penetration, but as complete knockdown was
caused at all dosages used, this explanation is unlikely.
Knockdown, or temporary incapacity, is exhibited by a number of
compounds, especially insecticides such as some organochlorines, carbamates,
and pyrethroids.[45] Members of this latter group that act rapidly are
considered to have good knockdown. Knockdown here is a prepoisoning
symptom and is mediated through action on the central or on the peripheral
nervous system. Structural variation within the pyrethroids influenced
knockdown, but the thiolactone kadethrin acted more rapidly than any other
pyrethroid, which usually lacks this moiety.[44] Whether these compounds act
through a similar mechanism to the pyrethroids, or even if the symptoms are

Bioactive Pseudo-C-nucleosides
285
related, is unclear, but their lack of lethal activity would indicate that another
process is operating. Additional support for this view comes from the distinct
structural differences between these nucleosides and the pyrethroids. This
effect is also similar to that of anaesthesia, such as is provided by carbon
dioxide in insects.
Enzyme inhibition
The activity of the two diastereoisomers 8 and 9 toward amphiphilic
acetylcholinesterase from human erythrocytes and butyrylcholinesterase
from human serum was investigated. The relative inhibition of these
enzymes has been found to exert a beneficial therapeutic effect in some
patients suffering from Alzheimer’s disease. Although in the assay acetylthio-
choline and butyrylthiocholine were used as substrates, it is considered that
they adequately represent the natural substrates for these hydrolytic
enzymes.[46] Enzyme activity was measured by the increase of yellow color
produced from thiocholine when it reacted with the dithiobisnitrobenzoate
ion. Inhibition of acetylcholinesterase activity at 1.7 mg/mL was 5% for 8
and 8% for 9. Inhibition of butyrylcholinesterase activity at 1.0 mg/mL was
36% for 8 and at 1.7 mg/mL was 22% for 9.
Conclusion
According to the data given in Table 2, the thiazole derivative 4 led to 100%
mortality of adult houseflies with dosages of 12 and 14 ng/insect and was the
only pseudo-nucleoside tested showing insecticidal activity. Regression
analysis on 24-hr mortality data for this compound based on percent mortality
and log dosage (Fig. 3) gave an estimate for the median lethal dosage
LD50 ¼ 6.7 ng/g (0.67 mg/g body weight), which is in the order of magnitude
of LD50 values for pyrethroids.
All three compounds exhibited knockdown at the concentrations used. No
estimate for ED50 for these compounds was found, but clearly it is less than
100 ng/insect. This property is clearly unusual and novel.
The thiazolidinone derivatives have demonstrated some efficacy for the
inhibition of butyrylcholinesterase (36% for 8 and 22% for 9). The enzyme
acetylcholinesterase was not significantly affected by these compounds (5%
for 8 and 8% for 9).
EXPERIMENTAL
General Methods
Melting points were determined by using either a Leitz-Biomed apparatus
or a Buchi 530. Optical rotations were determined with an Atago Polax-D

A. P. Rauter et al.
286
polarimeter, or in a Perkin Elmer 343. 1H NMR, 13C NMR, NOE, HMQC, and
COLOC experiments were recorded in CDCl3 either with a Bruker AC-P 250
(250 MHz) or with a Varian Unity-300 MHz, with Me4Si as internal standard.
Chemical shifts are expressed in ppm (d) and coupling constants J in Hz.
The 13C NMR spectra were recorded at 62.9 MHz.
IR spectra were recorded with a Biorad FTS (FT) or a Hitachi 270-50 spec-
trometer. Electron impact mass spectra were determined using a VG Trio 1000
spectrometer, by direct injection. Elemental analyses were performed with a
Fisons EA 1108 analyser.
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). Compounds were detected with UV light (254 nm) and/or by
spraying the sheets with a 3% vanillin-sulphuric acid solution. Column chrom-
atography (CC) was conducted under low pressure by elution from silica gel
(0.040–0.063 mm, Merck) columns.
Acetylcholinesterase from human erythrocytes and butyrylcholinesterase
from human serum were purchased from Sigma, Poole, UK.
Computational Details
DFT[47] calculations were performed using the Gaussian 98 program, rev.
A.11.[48] Molecular structures were fully optimized without any symmetry
constraints at the B3LYP[49] level with the 6-311Gꢀꢀ basis set[50] for all atoms.
Frequency calculations were performed to confirm the nature (minima) of
the stationary points determined. NMR chemical shifts were calculated
using the GIAO algorithm[51] and referenced to TMS (Tetramethylsilane).
Synthesis of Compounds 2–9
3-O-Benzyl-1,2-O-isopropylidene-a-D-ribo-pentofuranose-4-nitrile (2).
A solution of aldehyde 1 (7.25 mmol, 2 g) in pyridine (4 mL) was added slowly
to a solution of hydroxylamine hydrochloride (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 precipitated oxime. The mixture remained under stirring at rt for 1 hr.
CuSO4.5H2O was added (14.4 mmol, 3.6 g), followed by a solution of triethyl-
amine (14.4 mmol, 2 mL) in dichloromethane (3.6 mL) and DCC (8.72 mmol,
1.8 g) in dichloromethane (15 mL). After 2 hr at rt, formic acid was added
(1.3 mL) to eliminate DCC. The mixture was filtered, and the filtrate was con-
centrated under vacuum. The residue was treated with water (50 mL), 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 concen-
trated under vacuum to give 2 (1.54 g, 78%); m.p. 123–1248C; Rf ¼ 0.74 (ethyl
acetate/toluene 1:2); [a]D25 ¼ þ598 (c1.9, CHCl3); IR (KBr) (cm21): 2117
1
(CN); UV (lmax. nm) ethanol: 209 (1 ¼ 6887); H NMR (250 MHz): 7.32 (s, 5
´

Bioactive Pseudo-C-nucleosides
287
H, Ph), 5.74 (d, 1H, H-1, J1,2 ¼ 3.4), 4.78, 4.71 (AB system, OCH2, Bn), 4.62
(d, 1H, H-4, J3,4 ¼ 9), 4.53 (t, 1H, H-2, J2,3 ¼ 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).
Anal. Calcd for C15H17O4N: C, 65.44; H, 6.22; N 5.09. Found: C, 64.95; H,
6.38; N, 5.52.
3-O-Benzyl-1,2-O-isopropylidene-a-D-ribo-pentofuranose-4-thioamide
(3). DMAP (5.92 mmol, 0.072 g) was added to a solution of 2 (3.64 mmol, 1 g) in
chloroform (18 mL). Hydrogen sulfide was bubbled into the solution until full
saturation, and the reaction mixture was kept under stirring at 308C for 6 hr
30 min. After evaporation of the hydrogen sulfide that did not react, dichloro-
methane (50 mL) was added, and the solution was extracted with sodium
chloride solution 10% (w/v) (2 ꢂ 30 mL). The organic phase was dried
with magnesium sulfate, filtered, and concentrated in vacuum to give 3
(0.96 g, 85%); m.p. 194.3–194.68C; Rf ¼ 0.51 (ethyl acetate/toluene 1:1);
[a]D25 ¼ þ608 (c2.2, CHCl3); IR (KBr) (cm21): 3447, 3325 (NH2); UV (lmax. nm)
´
ethanol: 271 (1 ¼ 10292), 208 (1 ¼ 9372); 1H NMR (250 MHz): 7.77 (s, 1H,
NH2), 7.59 (s, 1H, NH2), 7.46–7.26 (m, 5 H, Ph), 5.80 (d, 1H, H-1, J1,2 ¼ 3.5),
4.87–4.74 (m, 3H, H-4, OCH2, Bn), 4.52 (t, 1H, H-2, J2,3 ¼ 4), 3.77 (dd, 1H,
H-3, J3,4 ¼ 8.6), 1.59 (s, 3H, CH3, isop), 1.37 (s, 3H, CH3, isop); 13C NMR:
203.71 (Cq, C-5), 136.22 (Cq, Ph), 128.34, 128.20, 128.13 (CH, Ph), 113.90
(Cq, isop), 103.43 (CH, C-1), 83.15 (CH, C-4), 81.96 (CH, C-3), 78.50 (CH,
C-2), 72.94 (OCH2, Bn), 26.96 (CH3, isop), 26.62 (CH3, isop).
Anal. Calcd for C15H19O4NS: C, 58.23; H, 6.19; N, 4.53; S, 10.36. Found: C,
57.85; H, 6.08; N, 4.52; S, 10.71.
Ethyl 2-[(4R)-3-O-benzyl-1,2-O-isopropylidene-a-D-erythrofuranos-4-
C-yl]thiazole-4-carboxylate (4). To a solution of ethyl bromopyruvate
(9.93 mmol, 1.24 mL) in anhydrous acetonitrile (6 mL) cooled at 08C, a
solution of 3 (3.24 mmol, 1 g) in anhydrous acetonitrile (15 mL) was added at
08C. The reaction mixture was ice-cooled. After 5 min stirring, the ice bath
was removed, and the solution was stirred at 208C for 1 hr. Evaporation of
the solvent under vacuum (208C) gave a residue, which was treated with a
solution of sodium hydrogen carbonate (100 mL) and extracted with dichloro-
methane (3 ꢂ 100 mL). The organic phase was washed with water and dried
with magnesium sulfate. The dichloromethane was evaporated under
vacuum to give a residue, which was purified by CC eluted with ethyl
acetate/toluene 1:5 (v/v) to give 4 (0.65 g, 50%) as a syrup; Rf ¼ 0.57 (ethyl
acetate/toluene 1:2); [a]D25 ¼ þ668 (c1.5, CHCl3); IR (neat) (cm21): 1741
(C55O), 1596 (C ¼ C); UV (lmax. nm) ethanol: 200 (1 ¼ 8950); 1H NMR
´

A. P. Rauter et al.
288
(250 MHz): 8.17 (s, 1H, H-5), 7.24 (s, 5 H, Ph), 5.88 (d, 1H, H-10, J1 ,2 ¼ 3.3), 5.41
0
0
(d, 1H, H-40, J3 ,4 ¼ 8.6), 4.74-4.59 (m, 3H, H-20, OCH2, Bn), 4.20 (q, 2 H, CH2,
0
0
Et, J ¼ 7.1), 3.99 (dd, 1H, H-30, J2 ,3 ¼ 4.1), 1.66 (s, 3H, CH3, isop), 1.43–1.36
(m, 6 H, CH3, isop, CH3, Et); 13C NMR: 169.08 (Cq, C55O), 161.43 (Cq, C-2),
147.50 (Cq, C-4), 137.01 (Cq, Ph), 128.29 (CH, C-5), 128.11, 128.03, 127.90
(CH, Ph), 113.69 (Cq, isop), 104.04 (CH, C-10), 82.55 (CH, C-30), 78.22 (CH,
C-20), 77.51 (CH, C-40), 72.36 (OCH2, Bn), 61.47 (OCH2, Et), 26.89 (CH3,
isop), 26.47 (CH3 isop), 14.34 (CH3, Et).
0
0
Anal. Calcd for C20H23O6NS: C, 59.25; H, 5.72; N, 3.45; S, 7.91. Found: C,
59.61; H, 5.79; N, 3.13; S, 7.73.
2-[(4R)-3-O-Benzyl-1,2-O-isopropylidene-a-D-erythrofuranos-4-C-yl]thia-
zole-4-carboxamide (5). A solution of 4 (2.47 mmol, 1 g) in anhydrous
methanol was cooled at 08C. Ammonia gas was bubbled into the solution
until complete saturation. After stirring for 48 hr at rt, the solvent was evapor-
ated to give 5 as a solid (0.79 g, 88%); m.p. 161–161.58C; Rf ¼ 0.43 (ethyl
acetate/toluene 2:1); [a]D25 ¼ þ408 (c1.25, CHCl3); IR (KBr) (cm21): 3480,
3405 (NH2), 1683 (C55O); UV (lmax. nm) ethanol: 233 (1 ¼ 11520), 219
´
1
(1 ¼ 11330); H NMR (250 MHz): 8.15 (s, 1H, H-5), 7.35–7.20 (m, 5 H, Ph),
5.91 (d, 1H, H-10, J1 ,2 ¼ 3.5), 5.82 (s, 1H, NH), 5.31 (d, 1H, H-40, J3 ,4 ¼ 8.8),
0
0
0
0
4.74–4.70 (m, 2H, H-20, part A of AB system, OCH2, Bn), 4.59 (1H, part B of
the AB system, OCH2, Bn, JAB ¼ 12), 3.92 (dd, 1H, H-30, J2 ,3 ¼ 4), 1.68
(s, 3H, CH3, isop), 1.42 (s, 3H, CH3, isop); 13C NMR: 168.39 (Cq, C55O),
162.75 (Cq, C-2), 149.40 (Cq, C-4), 136.78 (Cq, Ph), 128.51, 128.14, 127.89
(CH, Ph), 125.00 (CH, C-5), 113.80 (Cq, isop), 104.16 (CH, C-10), 82.56 (CH,
C-30), 77.88 (CH, C-20), 77.52 (CH, C-40), 76.48 (OCH2, Bn), 26.89 (CH3, isop),
26.47 (CH3 isop).
0
0
Anal. Calcd for C18H20O5N2S: C, 57.43; H, 5.35; N, 7.44; S, 8.52. Found: C,
57.12; H, 5.49; N, 7.88; S, 8.34.
5-[(4R)-3-O-Benzyl-1,2-O-isopropylidene-a-D-erythrofuranos-4-C-yl]tet-
razole (6). Ammonium chloride (4.6 mmol, 0.25 g) and sodium azide
(4 mmol, 0.342 g) were added to a solution of 2 (3.64 mmol, 1.0 g) in DMF
(8 mL). The mixture was heated at 1008C for 3 hr 30 min. The solution was con-
centrated, and the residue was extracted with ethyl acetate (3 ꢂ 50 mL) and
water (50 mL). The organic phase was washed with water and dried with mag-
nesium sulfate. The ethyl acetate was evaporated under vacuum to give 6
(1.23 g, 97%); mp. 149.8–150.18C; Rf ¼ 0.67 (ethyl acetate/methanol: 1/1);
[a]D25 ¼ þ698 (c1.8, CHCl3); IR (KBr) (cm21): 3480 (NH); UV (lmax. nm)
´
1
ethanol: 208 (1 ¼ 3390); H-NMR (250 MHz): 7.27 (s, 5 H, Ph), 5.93 (d, 1H,
H-10, J1 ,2 ¼ 3), 5.42 (d, 1H, H-40, J3 ,4 ¼ 8.8), 4.73–4.58 (m, 3H, H-20, OCH2,
0
0
0
0
Bn), 4.19 (dd, 1H, H-30, J2 ,3 ¼ 4.2), 1.62 (s, 3H, CH3, isop), 1.40 (s, 3H, CH3,
0
0
isop); 13C-NMR: 155.50 (Cq, C-5), 136.50 (Cq, Ph), 128.49, 128.28, 128.09

Bioactive Pseudo-C-nucleosides
289
(CH, Ph), 114.03 (Cq, isop), 104.57 (CH, C-10), 81.36 (CH, C-30), 77.79 (CH, C-20),
72.81 (OCH2, Bn), 71.48 (CH, C-40), 26.75 (CH3, isop), 26.36 (CH3, isop).
Anal. Calcd for C15H18O4N4: C, 56.60; H, 5.70; N, 17.60. Found: C, 56.86; H,
5.73; N, 17.21.
5-[(4R)-3-O-Benzyl-1,2-O-isopropylidene-a-D-erythrofuranos-4-C-yl]-5-
phenyl-1,2,4-triazole (7). Compound 3 (3.24 mmol, 1 g) was added to benzoyl
hydrazine (3.7 mmol, 0.5 g), and the mixture was heated at 1708C for 2 hr. After
cooling, the crude product was purified by CC eluted with ethyl acetate/toluene
1:5 (v/v) to give 7 as syrup (1.11 g, 87.5%); Rf ¼ 0.72 (ethyl acetate/toluene:
1/1). [a]2D5 ¼ þ788 (c1.6, CHCl3); IR (neat) (cm21): 3490 (NH2); UV (lmax. nm)
´
1
ethanol: 277 (1 ¼ 14600), 235 (1 ¼ 13350); H-NMR (250 MHz): 7.99 (dd, 2 H,
00 00
00 00
Ph-5, J2 ,3 ¼ 7.7, J2 ,4 ¼ 1.4), 7.54–7.49 (m, 3H, Ph-5), 7.21–7.15 (m, 5 H,
Ph, Bn), 5.88 (d, 1H, H-10, J1 ,2 ¼ 3.5), 5.29 (d, 1H, H-40, J3 ,4 ¼ 9), 4.76, 4.62
0
0
0
0
H, OCH2, Bn), 4.71 (t, 1H, H-20, J2 ,3
¼
0
0
(AB system,
2
4.2), 4.31 (dd, 1H, H-30), 1.69 (s, 3H, CH3, isop), 1.41 (s, 3H, CH3, isop);
13C-NMR: 165.54 (Cq, C-5), 163.16 (Cq, C-3), 136.69 (Cq Ph, Bn), 131.91,
128.92, 126.99 (3 CH, Ph-5), 128.37, 128.06, 127.89 (3 CH, Ph, Bn), 123.36
(Cq, Ph-5), 113.79 (Cq, isop), 104.38 (CH, C-10), 79.84 (CH, C-30), 77.58 (CH,
C-20), 72.65 (OCH2, Bn), 71.37 (CH, C-40), 26.85 (CH3, isop), 26.35 (CH3, isop).
Anal. Calcd for (C22H23O4N3): C, 67.16; H, 5.89; N, 10.68. Found: C, 67.52;
H, 6.45; N, 10.25.
(2S)-2-[(4R)-3-O-Benzyl-1,2-O-isopropylidene-a-D-erythrofuranos-4-C-
yl]-3-phenyl-2H-thiazolidin-4-one (8) and (2R)-2-[(4R)-3-O-Benzyl-1,2-
O-isopropylidene-a-D-erythrofuranos-4-C-yl]-3-phenyl-2H-thiazolidin-
4-one (9). To a solution of 1 (0.18 mmol, 0.05 g) in dry toluene (2 mL), activated
˚
powdered molecular sieves (4 A, 0.18 g), p-toluenesulfonic acid (catalytic
amount), and aniline (1.4 mmol, 0.13 g) in dry toluene (0.5 mL) were added.
The reaction mixture was refluxed under argon atmosphere with stirring, at
55–658C, in a reaction flask equipped with a Dean-Stark apparatus. After
3 hr, the mixture was cooled to rt, dry dichloromethane (20 mL) was added,
the mixture was filtered, and the filtrate was neutralized with a saturated
solution of sodium carbonate. The organic phase was dried with sodium
sulfate, filtered, and concentrated. Sulfanylacetic acid (0.628 mmol, 0.05 mL)
was added to the solution of the compound previously synthesized (0.111 g),
without purification, in dry toluene (15 mL). The mixture was heated at
55–658C under stirring in argon atmosphere, again with a Dean-Stark
apparatus coupled to the reaction flask. The mixture was cooled to rt after
50 hr 20 min heating, and dry toluene (15 mL) was added. After extraction
with water, the organic phase was dried over sodium sulfate, filtered, and con-
centrated. The isomers 8 and 9 were purified by CC eluted with ethyl acetate/
toluene 1:5 (v/v), to give 8 (0.044 g, 57 %) as a syrup and 9 (0.010 g, 13%).

A. P. Rauter et al.
290
Data for 8: Rf ¼ 0.69 (ethyl acetate/toluene 1:1); [a]2D0 ¼ 218 (c 0.05,
1
CHCl3); H NMR (300 MHz): 7.38–7.31 (m, 10H, 2Ph), 5.71 (d, 1H, H-10,
0
0
0
J1 ,2 ¼ 3.6), 5.37 (t, 1H, H-2, J2,4 ¼ 1.8), 4.65, 4.61 (1H, part A of AB system,
OCH2, Bn, JA,B ¼ 11.7), 4.56 (t, 1H, H-20, J2 ,3 ¼ 4.5), 4.36–4.28 (m, 2H, H-40,
0
0
of AB system, OCH2, Bn,), 3.91 (dd, 1H, H-30, J3 ,4
¼
0
0
part
B
8.4), 3.48, 3.42 (each s, 1H, H-5a, part A of AB system, J5a,5b ¼ 15.6), 3.36,
3.31 (each d, 1H, H-5b, part B of AB system, J2,5b ¼ 1.5), 1.45 (s, 3H, CH3,
isop), 1.31 (s, 3H, CH3, isop); 13C NMR: 170.8 (C55O); 137.8, 136.7 (Cq, Ph),
129.4, 128.6, 128.3, 127.7, 126.5, 125.5 (CH, Ph), 113.5 (Cq, isop), 104.1 (CH,
C-10), 79.3 (CH, C-40), 77.6 (CH, C-20), 77.1 (CH, C-30), 72.0 (OCH2, Bn), 63.8
(CH, C-2); 32.4 (CH2, C-5), 26.8 (CH3, isop), 26.6 (CH3, isop).
Anal. Calcd for C23H25NO5S: C, 64.62; H, 5.89; N, 3.28; S, 7.50. Found: C,
64.92; H, 5.79; N, 3.35; S, 7.62.
Data for 9: Rf ¼ 0.59 (ethyl acetate/toluene 1:2); [a]2D0 ¼ þ88 (c0.05,
1
CHCl3); H NMR (300 MHz): 7.42–7.24 (m, 10H, 2Ph), 5.73 (d, 1H, H-10, J1 ,2
¼
0
0
0
3.3), 4.85 (t, 1H, H-2, J2,4 ¼ 1.5, J2,5a ¼ 1.5), 4.72, 4.68 (1H, part A of AB
system, OCH2, Bn), 4.56 (t, 1H, H-20, J2 ,3 ¼ 3.9), 4.43, 4.39 (1H, part B of
0
0
AB system, OCH2, Bn, JA,B ¼ 12.3), 4.18 (dd, 1H, H-40, J3 ,4 ¼ 9.0), 3.81–3.72
(m, 2H, H-30, H-5a, part A of AB system), 3.43–3.38 (each s, 1H, H-5b, part
B of AB system, J5a,5b ¼ 15.3), 1.37 (s, 3H, CH3, isop), 1.32 (s, 3H, CH3, isop);
13C NMR: 171.1 (C55O), 137.0 (Cq, Ph), 129.6, 129.0, 128.8, 128.5, 128.2,
127.3 (CH, Ph), 113.8 (Cq, isop), 104.2 (CH, C-10), 78.5 (CH, C-30), 77.9 (CH,
C-20), 76.6 (CH, C-40), 72.4 (OCH2, Bn), 0.65.3 (CH, C-2), 33.1 (CH2, C-5),
26.9 (CH3, isop), 26.7 (CH3, isop).
0
0
Anal. Calcd for C23H25NO5S: C, 64.62; H, 5.89; N, 3.28; S, 7.50. Found: C,
64.78; H, 5.73; N, 3.44; S, 7.28.
Biological Assays
Aquatic brine shrimp bioassay
Newly hatched brine shrimp larvae, Artemia salina, were bioassayed using
a method based on microtitre plates.[52] Brine shrimp larvae were reared from
eggs in brine (15 g/L sodium chloride) in a small glass tank at 308C. Using their
phototropic response the emerging larvae were separated from the eggs.
Larvae up to 24 hr old only were used. Using a dissecting microscope the
larvae were carefully transferred (in 75 mL), in batches of five, using a piston
micropipette from the tank to the wells of the microtitre plate (each well
volume 300 mL). Solutions of the test substances in saline were then added to
the wells, and the plate was covered and kept at 308C. Eight replicates for
each concentration of each test compound and untreated control were used.
Percent mortality (no movement or response to a probe) was determined at
24 hr.

Bioactive Pseudo-C-nucleosides
291
Housefly bioassay
A pyrethroid susceptible strain (Cooper) of the housefly Musca domestica
was cultured at 258C.[53] This method included feeding the adults on milk
with 5% sucrose and egg laying and larval rearing on moist bran. Three-
day-old adults were selected (both sexes) and anaesthetized with carbon
dioxide. Each compound was applied to the ventral thorax in 1.0 mL acetone
using a microapplicator. Five insects (mean adult fly weight was 10 mg) were
used for each dosage of each compound. The responses were determined con-
tinuously up to 1 hr after treatment; recovery from anaesthesia, knockdown
(inability to recover normal posture). Final mortality was determined at
24 hr after treatment, keeping the temperature at 258C and food available.
Inhibition of Acetylcholinesterase and of Butyrylcholinesterase[54]
Incubation time (with inhibitor): 15 min at 308C + 1.
Reaction time: 6 min at 308C + 1.
In each well of a microtiter plate, enzyme solution (5 mL), pH 8 buffer
(200 mL), dithiobisnitrobenzoate (DTNB) reagent (5 mL), and the solution of
the tested compounds (5 mL) were placed. The solution of amphiphilic acetyl-
cholinesterase from human erythrocytes was used with the concentration
1.26 mg enzyme/mL pH 8 buffer, the solution of butyrylcholinesterase from
human serum had a concentration of 0.1 mg/mL, DTNB had a concentration
of 5.2 mg/mL pH 7 buffer, and the tested compounds were used with the
concentration of 1.7 mg/mL ethanol 86%, except when the inhibition of
butyrylcholinesterase was tested with compound 8, whose solution had the
concentration of 1.0 mg/mL ethanol 86%. After 15 min incubation the substrate
(5 mL), which was acetylthiocholine (6.4 mg/mL water) or butyrylthiocholine
(7 mg/mL water), was added. The reaction time was 6 min, and during this
time the reaction kinetics were measured by a UV spectrophotometer at
405 nm with a temperature control of 308C + 1. This procedure took place six
times. The three blank assays for such run consisted of buffer, DTNB, and
substrate. The enzyme solution was replaced by the same amount of water,
and the solution of compounds 8 or 9 was replaced by ethanol 86%.
There were also six control assays with the enzyme, buffer, and DTNB,
adding ethanol 86% to replace the solution of the tested compounds.
ACKNOWLEDGMENT
´
ˆ
The authors thank The British Council and the Ministerio da Ciencia e do
ˆ
Ensino Superior for financial support and Fundac¸a˜o para a Ciencia e Tecnologia
for a postdoctoral grant.

A. P. Rauter et al.
292
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