Synthesis and nematocide activity of S-glycopyranosyl-6,7-diarylthiolumazines.

Article abstract of DOI:10.1016/j.bmc.2004.06.004

6,7-Diaryl derivatives of mono and di-S-glycopyranosylthiolumazine derivatives 5-8 were prepared to test their nematocide activity. In vitro tests against Caenorhabditis elegans were performed and it was found that monosubstituted derivatives 5-7 showed higher activity than the corresponding unsubstituted 2-thiolumazines 1-3, whilst 2-S,4-S-di-glycopyranosylpteridine derivative 8 was inactive in contrast to unsubstituted derivative 4. In order to check whether the lack of activity of 8 was due to the two bulky substituents of the pteridine nucleus, 2-S,4-S-dimethyl derivative 9 was synthesized and assayed showing also lack of activity. A theoretical study on the stability of the different possible tautomers of compound 4 was carried out in an attempt to explain some, in appearance, anomalous (13)C NMR data of this compound.

Full text of DOI:10.1016/j.bmc.2004.06.004

Bioorganic & Medicinal Chemistry 12 (2004) 4431–4437
Synthesis and nematocide activity
of S-glycopyranosyl-6,7-diarylthiolumazines
Miriam A. Martins Alho,a Norma B. D’Accorso,a Carmen Ochoa,b,* Ana Castro,b
b
Felix Calderon, Antonio Chana, Felipe Reviriego, Juan Antonio Paez,
b
b
b
ꢀ
ꢀ
ꢀ
Nuria E. Campillo, Mercedes Martınez-Grueiro, Ana Marıa Lopez-Santa Cruz
b
c
c
ꢀ
and Antonio R. Martınez
ꢀ
ꢀ
c
ꢀ
a
ꢀ
ꢀ
Centro de Investigaciones en Hidratos de Carbono, Departamento de Quımica Organica,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
bInstituto de Quꢀımica Mꢀedica (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
c
ꢀ
Departamento de Parasitologıa, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
Received 27 February 2004; accepted 2 June 2004
Available online 2 July 2004
Dedicated to the memory of Dr. Manfred Stud
Abstract—6,7-Diaryl derivatives of mono and di-S-glycopyranosylthiolumazine derivatives 5–8 were prepared to test their nema-
tocide activity. In vitro tests against Caenorhabditis elegans were performed and it was found that monosubstituted derivatives 5–7
showed higher activity than the corresponding unsubstituted 2-thiolumazines 1–3, whilst 2-S,4-S-di-glycopyranosylpteridine
derivative 8 was inactive in contrast to unsubstituted derivative 4. In order to check whether the lack of activity of 8 was due to the
two bulky substituents of the pteridine nucleus, 2-S,4-S-dimethyl derivative 9 was synthesized and assayed showing also lack of
activity. A theoretical study on the stability of the different possible tautomers of compound 4 was carried out in an attempt to
explain some, in appearance, anomalous 13C NMR data of this compound.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
control of human helminths.3 Despite the availability of
highly effective, broad-spectrum agents, there is always
an urgent need for safer, more convenient and more
environmentally friendly products that will overcome
the ever present threat of resistance development.4 A
prioritized list of research objectives in veterinary par-
asitology includes the need of new drugs.5 In spite of this
recommendation, not many articles on this topic have
appeared recently.
It is estimated that about 1.3–2.0 billion people in the
world suffer from helminth infections. Although direct
mortality is low, nematode parasites cause serious
morbidity mainly in the ‘Third World’. The infections in
humans and livestock are controlled by anthelmintic
drugs, but the chemotherapy of many helminth infec-
tions is complicated by the widespread development of
drug resistance to most of the broad-spectrum drugs in
many parasite species of livestock animals.1;2 Further-
more, there is an increased awareness of the potential
problem of anthelmintic resistance in the treatment and
In previous papers, we have described the synthesis and
nematocide activity of several 6,7-diarylpteridines
bearing different substituents, none of them being a
glycosidic moiety.6–8 Many of these compounds showed
interesting nematocide properties, and structure–activity
relationship studies, using the Comparative Molecular
Field Analysis (CoMFA)9 and neural networks tech-
nique,7 were carried out. It was observed that N-meth-
ylation decreased the nematocide activity whilst
S-methylation increased it.6;7 In order to investigate the
effect of a carbohydrate substituent on the nematocide
Keywords: Synthesis; 13C NMR; S-Glycopyranosylthiolumazines;
Tautomerism; Nematocide properties; Caenorhabditis elegans.
* Corresponding author. Tel.: +34-915622900; fax: +34-915644853;
e-mail: carmela@iqm.csic.es
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.004

4432
M. A. Martins Alho et al. / Bioorg. Med. Chem. 12 (2004) 4431–4437
p-dimethoxybenzil following the usual method to obtain
O
O
pteridines.12 S-Methylation of 4 using tetrabutylammo-
nium fluoride (TBAF), as base, and an excess of
methyl iodide, as alkylating agent, afforded dimethyl
derivative 9 (see Scheme 2). Attempts to obtain the
corresponding S-monomethyl derivatives were unsuc-
cessful due to the similar reactivity against alkylating
agents of both thioxo groups. So, when equimolar
amounts of alkylating agent and pteridine 4 were used,
only a complex and inseparable mixture, probably of 2-
S- and 4-S-monomethylated derivatives, S,S0-dimelth-
ylated derivative and starting material was obtained.
An excess of alkylating agent afforded S,S0-dimethyl
derivative.
N
N
N
N
R1
R1
R1
R1
HN
R2S
HN
N
S
N
H
5
6
7
R1
R1
R1
=
=
=
=
phenyl,
1, R1 = phenyl
2, R1 = 2-thienyl
3, R1 =p-methoxyphenyl
2-thienyl
p-methoxyphenyl
6-(1,2:3,4-di-O-isopropylidene-
α-D-galactopyranosyl)
5, 6, 7, R2
Scheme 1. Structure of pteridine derivatives 1–3 and 5–7 previously
synthesized.
activity of pteridines, monosubstituted 2-S-(6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranos-6-yl) de-
A previous report on the synthesis of S-, N- and
O-glycosides derived of compound 1 had appeared in
the literature.14 6,7-Diaryl-2-[(60-deoxy-10,20:30,40-di-O-
rivatives (5–7) of 6,7-diaryl-2-thiolumazines (1–3)
(Scheme 1) had previously been synthesized.10 To check
the effect of the introduction of a second glycosidic
substituent on the nematocide activity, the synthesis of
the unknown 6,7-bis(p-methoxyphenyl)-2,4-pteridine-
dithione 4 and its corresponding 2-S,4-S-bis(6-deoxy-
isopropylidene-a-D
-galactopyranos-60-yl)thio]pteridin-
4-one derivatives 5–7 were previously prepared by us
from 6,7-diaryl-2-thiolumazines 1–3, respectively.10 In
agreement with previous results15 in the synthesis of
1,2:3,4-di-O-isopropylidene-a-D-galactopyranos-6-yl)
2-[(60-deoxy-10,20:30,40-di-O-isopropylidene-a-
D-galacto-
derivative 8 has now been performed. S,S0-Dimethyl
substituted derivative 9 has also been synthesized in
order to check if the potential nematocide property of 8
could be mediated by bulky substituents such as car-
bohydrate moieties. In this work, the nematocide
activity of pteridines bearing carbohydrate moieties 5–8,
as well as, pteridines 4 and 9 has been evaluated using
Caenorhabditis elegans as model. C. elegans has become
a popular model system for searching nematocide
activity, since it is easy to maintain and has a very fast
life-cycle.11
pyran-60-yl)thio]-4-hydroxypyrimidine from 4-hydroxy-
2-mercaptopyrimidine, these kind of heterocycles can
generate an S-anion by aromatization of the pyrimidine
ring, and acting as a nucleophile replacing a good
leaving group. Thus, S-glycosylation of 1–3 using 6-O-
tosyl-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
as glycosylating agent and sodium ethoxide as strong
base afforded derivatives 5–7. To obtain the S,S0-
disubstituted derivative 8, from compound 4, identical
reaction conditions were attempted. Results, under these
conditions, were unsuccessful, since, only decomposition
of starting base was observed. Glycosylation reaction of
4 was finally achieved by using an excess of 6-iodo-6-
2. Results and discussion
2.1. Chemistry
deoxy-1,2:3,4-di-O-isopropyliden-a-D-galactopyranose
instead the corresponding tosyl derivative (see Scheme
3). S,S0-Diglycopyranosyl derivative 8 was obtained in
similar yield as compounds 5–7. The 1H NMR spectrum
of derivative 8 is very simple for a disubstituted deriv-
ative. However, 13C NMR data (Table 2) of C-2 and C-4
are in agreement with a double substitution and are
comparable to those of dimethyl derivative 9. On the
other hand, analysis of compound 8 using FAB-MS
shows a peak (892) corresponding to the molecular
weight.
6,7-Diaryl-2(1H)-thioxo-4(3H)-pteridinones 1 and 26
and 6,7-bis(p-methoxyphenyl)-2(1H)-thioxo-4(3H)-pte-
ridinone 37 had previously been synthesized by us
following two different methods.12;13 6,7-Bis-(p-methoxy-
phenyl)-2,4(1H,3H)-pteridinedithione 4 has now been
synthesized, in moderate yield (45%), by condensation
between 4,5-diamino-2,6-dimercaptopyrimidine and
S
S
N
O
O
OMe
OMe
NH2
NH2
OMe
HN
EtOH/H+
reflux
HN
+
S
N
H
N
OMe
S
N
H
4
S
SMe
N
N
N
N
N
OMe
OMe
OMe
OMe
(Bu)4N+F-, THF
MeI
HN
N
S
N
H
MeS
9
4
Scheme 2. Synthetic pathway of compounds 4 and 9.

M. A. Martins Alho et al. / Bioorg. Med. Chem. 12 (2004) 4431–4437
4433
I
S
S -
N
O
N
N
Na+EtO-
EtOH
R1
R1
N
N
R1
R1
DMF
HN
N
3
O
O
+
reflux
S
N
H
- S
O
O
4
O
O
S
N
O
O
N
R1
R1
N
R1
=
O
S
N
OMe
O
O
O
O
O
8
Scheme 3. Synthetic pathway of compound 8.
Table 1. 13C NMR chemical shiftsa of pteridine derivatives 1–9
Compound C-2
C-4
C-4a
C-6
C-7
C-8a
Carbon of substituents other than pteridine nucleus
1b;6
2b;6
3b;7
4b
179.2
162.6
157.9
159.2
187.3
160.5ꢀ
121.0
126.4
126.6
129.6
128.9
146.0
141.5
147.0
142.8
151.9
157.3
149.0
156.1
156.0
158.3
148.3
146.6
149.6
149.9
152.8
137.8, 137.6, 130.2, 130.0, 129.1, 128.3
175.7
175.8
172.8
161.1ꢀ
139.5, 139.2, 132.5, 130.7, 129.3, 128.9, 127.7
160.1, 161.1, 132.0, 131.4, 130.7, 129.8, 114.5, 114.3, 55.8
161.1, 160.1, 131.7, 131.3, 130.8, 130.6, 114.2, 55.6
5c;10
137.6, 129.9, 129.8, 129.6, 129.3, 128.1, 109.6, 108.9, 96.4, 71.8,
70.8, 70.5, 66.6, 31.7, 26.0–24.3
6c;10
7c;10
8c
161.5ꢀ
160.8ꢀ
169.1ꢀ
159.8ꢀ
160.2ꢀ
173.3ꢀ
129.2
128.7
128.3
143.3
151.3
149.3
152.2
159.9
158.7
150.1
152.5
150.4
140.3, 139.9, 131.8, 130.4, 129.7, 128.3, 127.9, 109.7, 109.1,
95.6, 71.8, 70.9, 70.6, 66.8, 31.8, 26.1–24.4
159.9, 131.5, 131.2, 131.3, 130.1, 113.6, 109.5, 108.8, 96.3, 71.7,
70.8, 70.5, 66.5, 55.1, 31.5, 25.9–24.3
160.1, 159.6, 130.7, 130.4, 112.8, 112.7, 109.5, 108.8, 95.6, 70.9,
70.6, 69.9, 69.5, 65.5, 65.1, 54.3, 30.4, 28.6, 25.9, 25.0, 24.8
161.5, 161.0, 132.1, 131.8, 130.8, 114.2, 114.1, 55.7, 15.3, 13.0
160.6, 160.1, 131.5, 131.1, 130.1, 113.8, 113.7, 55.3, 14.3, 12.2
9c
9b
172.0ꢀ
170.3ꢀ
175.1ꢀ
174.3ꢀ
129.8
128.7
150.6
149.7
160.1
159.4
151.8
151.1
a d in ppm.
b DMSO-d6 as solvent.
c CDCl3 as solvent.
* Assignments could be interchanged.
Table 2. Calculated energies (E, hartrees), relative energies (DE, kcal molꢁ1) in gas phase and in the COSMO model, and dipole moments (l, Debye)
of tautomers 12A–12B
Method
12A
DE
12B
DE
E
l
E
l
Vacuum
Vacuum
CHCl3
H2O
B3LYP/6-31G*
DMol
)1246.450073
)1240.132822
)1240.146679
)1240.153048
0.00
0.00
0.00
0.00
4.50
3.58
––
)1246.42598
)1240.108598
)1240.124451
)1240.131829
15.12
15.20
13.95
13.31
2.77
2.28
––
COSMO/DMol
COSMO/DMol
––
––
In this case preparation of S-monosubstituted deriva-
tives was also unsuccessful and only inseparable com-
plex mixtures were obtained when glycosylating agent
and starting base were used in equimolar amount.
Galactopyranose iodide was obtained by treatment of
the protected galactose with I2/Ph3P.16
2.2. 13C NMR study
In order to compare 13C NMR data, 13C chemical shifts
of new compounds 4, 8 and 9 together with those pre-
viously reported for compounds 1–3, 5–7 are shown in
Table 1. The NMR study of compound 4 was carried

4434
M. A. Martins Alho et al. / Bioorg. Med. Chem. 12 (2004) 4431–4437
157.9
185.9
N
186.2
N
O
4
S
S
R1
R1
R1
R1
R1
R1
N
4a
6
HN
HN
HN
2
8a
7
S
N
H
N
S
O
N
H
N
N
H
N
175.7
150.1
175.6
2, R1 = 2-thienyl
10, R1 = 2-thienyl
11, R1 = 2-thienyl
Scheme 4. C-2 and C-4 Chemical shifts of oxo and thioxo pteridines 2, 10 and 11.
out in DMSO-d6 solution because this compound is very
insoluble in CDCl3. 13C chemical shifts of compound 4
were assigned by comparing the chemical shifts of the
corresponding 2-thioxo,4-oxo-derivatives 1–3. On the
other hand, the correct assignment for C-2 and C-4 was
checked by comparison with the corresponding chemical
shifts of related derivatives 2, 10 and 11 previously
reported6 (see Scheme 4).
stable tautomer and form B as the tautomer compatible
with 13C NMR data. Thus, the study in gas phase was
performed using two density functional methods, a
B3LYP/6-31G*17;18 and a local density functional
method (LDF), which include electron correlation19–21
with DMol program.22 Results obtained indicate that in
)
than tautomer B (Table 2) and therefore, the form
present in the gas phase.
the gas phase tautomer A is more stable (15 kcal molꢁ1
As can be seen in Table 1 and Scheme 4, the chemical
shift of C-4 is shifted downfield about 30 ppm by
changing an oxo group (compounds 2 and 3) by a thioxo
group (compounds 4 and 11 about 187 ppm), whilst C-2
chemical shift’s changes from 150 ppm to about 175 ppm
when an oxo is transformed into a thioxo group
(Scheme 4).
To study the relative stability of the two tautomers in
solution, the solvation effect has been considered via the
COSMO method23;24 implemented in DMol. The Con-
ductor-like Screening Model (COSMO) is a continuum
solvation model, where the solute molecule forms a
cavity within the dielectric continuum of permittivity e
that represents the solvent.
NMR spectra of soluble compounds in CDCl3 (5–9)
were recorded in this solvent. In order to compare 13C
chemical shifts of compounds 4 and 9, the 13C NMR
spectrum of 9 was also recorded in DMSO-d6 showing
both spectra only slight chemical shift differences.
Comparing 13C NMR spectra of unsubstituted deriva-
tive 4 and S,S0-dimethyl derivative 9 (recorded both in
DMSO-d6), it is interesting to point out whilst the C-4
signal appears shifted 13 ppm to upfield in 9, as expected
for the change of an N–C@S to N@C–S-group (com-
pare C-2 chemical shifts of 1–3 to those 5–7), the signal
corresponding to C-2 only changes slightly (172.8–
170.3 ppm). One possible explanation, of this anomalous
fact, could be the existence of compound 4 mainly in the
form of tautomer B, in DMSO solution (see Scheme 5).
The results obtained with COSMO model in water or in
chloroform, collected in Table 2, indicate that tautomer
A is more stable than tautomer B, 13 or 14 kcal molꢁ1, in
water or chloroform, respectively. The theoretical re-
sults in solution from COSMO model provide a stabil-
ization of tautomer B, about 2 kcal molꢁ1 in relation to
gas phase; however, the large stability difference of
tautomer A in relation to B indicates that only form A
should be present in chloroform or water. On the other
hand, the value of dipole moments in vacuum indicates
that tautomer A would be more favoured than tautomer
B in polar solvents.
2.4. Biological study
2.3. Theoretical study on tautomerism
The in vitro nematocide activity of compounds 4–9 was
tested against larvae of C. elegans. The use of C. elegans,
a free-living nematode, for the in vitro test is a safe and
fast method to evaluate the nematocide activity of
compounds.25 In order to compare with the data of
pteridines previously evaluated (1–3),6;7 the same meth-
od and conditions were used. All compounds were tested
at concentration of 100 lg/mL, subsequently, com-
In view of these results, we decided to study the tauto-
merism in a more simple pteridine, 12, in gas phase and
in solution by theoretical molecular orbital calculations.
In principle, 2,4-(1H,3H)-pteridinedithione 12 can exist
as six possible tautomers, however, only tautomers A
and B were calculated, form A as the probably more
S
4
SR
187.3
N
S
N
2 174.3*
N
187.3
N
R1
R1
R1
R1
R1
R1
HN
2
HN
DMSO
N
MeI
S
N
H
N
HS
172.8
N
R2S
170.3*
N
N
172.8
4A, R1 = p-OMePh
12A, R1= H
4B, R1 = p-OMePh
12B, R1= H
9, R1 = p-OMePh, R2 = Me
Scheme 5. Tautomers A and B of 4 and 12 and C-2, C-4 data of 4 and 9 in DMSO solution.

M. A. Martins Alho et al. / Bioorg. Med. Chem. 12 (2004) 4431–4437
4435
Table 3. Nematocide in vitro activity of compounds 1–9 against
C. elegans
methyl, 2-S-methyl-substituded derivatives of some 6,7-
diarylthiolumazines, without acid protons, showed
nematocide activity.6;7 On the other hand, the inactivity
of S,S0-diglycosyl derivative 8 could not be due to its
excessive bulkiness because the S,S0-dimethyl derivative
9 is also inactive, unless, one admits that the steric
hindrance of S-methyl group at C-4 position should be
enough to diminish nematocide activity.
Compd
IC50 (lg/mL)
IC50 (mM)
16
26
37
4
50
1.72 · 10ꢁ1
>2.91 · 10ꢁ1
6.38 · 10ꢁ2
6.13 · 10ꢁ2
4.18 · 10ꢁ2
1.99 · 10ꢁ2
2.37 · 10ꢁ2
>1.12 · 10ꢁ1
>2.29 · 10ꢁ1
2.67 · 10ꢁ3
>100
25
25
5
24
10
6
7
15
8
>100
>100
1–0.5
9
Mb
4. Experimental section
4.1. Chemistry
pounds showing activity higher than 50% were tested at
lower concentration (50, 25, 10, 2 and 0.5 lg/mL) to
determine the inhibition concentration required to
obtain up to 50% (IC50) in the reduction of population
growth of nematode.
Melting points were determined with a Kofler hot-stage
apparatus and are uncorrected. Flash column chroma-
tography was performed on Merck silica gel (230–400
mesh). Analytical TLC was performed on aluminium
sheets coated with 0.2 mm layer of silica gel 60 F254. 1H
and 13C NMR were recorded on a Varian Gemini-200 at
200 and 50 MHz, respectively, for compounds 4 and 9,
and on a Bruker-500 at 500 and 175 MHz, respectively,
for compound 8, using tetramethylsilane (TMS) as
internal standard. MS of compound 8 was registered by
fast atomic bombardment (FAB) technique on a ZAB-
SEQ4F spectrometer, using glycerol matrix.
Compounds 1, 3–7 showed more than 90% of reduction
in the nematode population growth at the higher con-
centration tested (100 lg/mL); thus, these compounds
present remarkable nematocide activity at this concen-
tration. However, compound 8 exhibited only 40% of
reduction and compounds 2 and 9 no reduction at this
concentration. The nematocide activity of compounds 1,
3–7 were assayed at lower concentrations and their IC50
in lg/mL and mM concentrations are gathered in Table
3 together with those corresponding to mebendazole
(Mb) used as standard. The most active pteridines, now
tested, are the S-mono-glycosyl derivatives 6 and 7.
S-Mono-glycosyl derivatives present higher nematocide
activity than the corresponding unsubstituted deriva-
tives (comparing IC50 of 1 and 5, 2 and 6 and 3 and 7 in
Table 3) mainly in the case of 6,7-dithienyl derivative 2.
Substitution of 4-oxo by 4-thioxo group did not modify
the activity (comparing 3 and 4). When a second sub-
stituent, S-glycosyl or S-methyl, is introduced in the 4
position of pteridine the activity is completely lost
(comparing 7 to 8 and 9).
4.2. 6,7-Bis(p-methoxyphenyl)-2,4(1H,3H)-pteridinedi-
thione 4
To a solution of 4,5-diamino-2,6-dimercaptopyrimidine
(0.87 g, 5 mmol) in ethanol (50 mL), p-dimethoxybenzyl
(1.35 g, 5 mmol) and some drops of hydrochloric acid
were added under reflux. The solutions was kept on
refluxing for 6 h. Then, the reaction mixture was con-
centrated and the solid obtained suspended in hexane
(100 mL). The suspension was heated under reflux over
30 min in order to remove the excess of a-dicarbonylic
compound. The remaining solid was filtered and purified
by column chromatography using CH2Cl2/CH3OH (30/
1 v/v) as eluent. Compound 4 (0.94 g) was obtained in
1
45% yield: mp ¼ 283–285 ꢁC (CH3OH/H2O). H NMR
1
3. Conclusions
(DMSO-d6) d (ppm): 13.84 (br s, H), 13.75 (br s, 1H),
7.44 (d, 3J ¼ 8:8 Hz, 2H), 7.36 (d, 3J ¼ 8:8 Hz, 2H), 6.95
3
3
Results of theoretical calculations showed that the most
stable tautomer in the gas phase is form A. Although,
results in solution indicated a slight stabilization of
tautomer B, the most stable tautomer is still tautomer A.
(d, J ¼ 8:8 Hz, 2H), 6.93 (d, J ¼ 8:8 Hz, 2H) 3.78 (s,
6H). Anal. Calcd for C20H16N4O2S2: C, 58.82; H, 3.92;
N, 13.72; S, 15.68. Found: C, 59.10; H, 4.07; N, 14.02; S,
16.00.
Except for derivatives 2, 8 and 9, the pteridines studied
exhibited nematocide activity, although none of them
reached the activity of mebendazole used as standard.
Substitution at 2-S-position enhances nematocide
activity, whilst disubstitution at 2-S,4-S-positions elim-
inates this activity. We have not found the reason to
explain the lack of activity of the S,S0-unsubstituted-6,7-
dithienyl derivative 2 in contrast to the activity of
compounds 1, 3 and 6.
4.3. 2,4-Bis[(60-deoxy-10,20:30,40-di-O-isopropylidene-a-
D-
galactopyranos-60-yl)thio]-6,7-bis(p-methoxypheyl)pter-
idine 8
6,7-Di-(p-methoxyphenyl)-2,4-pteridinedithione (230 mg,
0.56 mmol) were placed into a 25 mL round flask,
and 1.7 mL of a solution of sodium ethoxide were
added (the later solution was prepared by dissolution of
190 mg on metallic sodium in 20 mL of ethanol). The
suspension was stirred in a warm bath (45 ꢁC), then
evaporated to dryness and to this solid, 557 mg
The inactivity of compounds 8 and 9 could be due to the
absence of acid protons in the molecule; however, 1-N-

4436
M. A. Martins Alho et al. / Bioorg. Med. Chem. 12 (2004) 4431–4437
(1.5 mmol) of 6-deoxy-6-iodo-1,2:3,4-di-O-isopropylid-
-galactopyranose (synthesized as it was de-
4.6. Nematocide activity test
ene-a-
D
scribed in literature16) and 10 mL of DMF, were added.
The mixture was heated to reflux until no changes were
detected by TLC (approximately 4 h). The mixture was
concentrated under reduced pressure, purified by flash
chromatography using cyclohexane/acetone (9:1 v/v) as
eluent and the title compound was obtained as a syrup
The compounds were tested for nematocide activity
against the free-living nematode C. elegans following the
method of Simpkin and Coles25 with slight modifica-
tions.26 Tests were carried out in 24-well plates (Co-
starꢂ) and four wells were used for each experimental
group. To each well, 1 mL of culture medium was added
followed by 5 lL of the appropriate compound solution.
The products were dissolved in dimethylsulfoxide and
the same concentration of solvent (0.5%) was incorpo-
rated to control wells. Finally, 0.5 mL of culture medium
containing 10–15 C. elegans larvae (L2 or L3 obtained
of synchronous cultures) was added to each well.
1
(208 mg, 40.8% yield). H NMR (CDCl3) d (ppm): 7.58
3
3
(d, J ¼ 8:7 Hz, 2H), 7.54 (d, J ¼ 8:7 Hz, 2H), 6.85 (d,
3J ¼ 8:7 Hz, 4H), 5.57 (d, J ¼ 4:7 Hz, 2H), 4.31 (dd,
3
3J ¼ 4:7 Hz, 3J ¼ 2:6 Hz, 2H), 4.65 (dd, 3J ¼ 7:5 Hz,
3J ¼ 2:6 Hz, 1H), 4.63 (dd, 3J ¼ 7:5 Hz, 3J ¼ 2:6 Hz,
1H), 4.45 (m, 2H), 4.23 (m, 1H), 4.16 (m, 1H), 3.84 (s,
6H), 3.63 (dd, 2J ¼ 13:9 Hz, 3J ¼ 6:2 Hz), 3.52 (dd,
2J ¼ 13:9 Hz, 3J ¼ 7:7 Hz), 1.48–1.29 (12H). MS m=z:
892. (M). Anal. Calcd for C44H52N4O12S2: C, 59.19; H,
5.38; N, 6.28; S, 7.77. Found: C, 59.33; H, 5.69; N, 6.15;
S, 7.60.
The effect of compounds on the development and
reproductive capacity of C. elegans was determined by
comparing the populations levels attained in the control
and test wells after an incubation period of 7 days at
20 1 ꢁC. Inhibition concentration (IC50) was calculated
from the reduction percentage of nematode population
growth at different doses of test compounds in relation
with controls.
4.4. 6,7-Bis(p-methoxyphenyl)-2,4-bis(methylthio)-
pteridine 9
To a solution of 2,4-pteridinedithione 4 (0.82 g, 2 mmol),
in tetrahydrofurane (14 mL) containing tetrabutyl-
ammonium fluoride (THF/TBAF 1/5), methyl iodide
(1.15 g, 8 mmol) was added dropwise. The reaction
mixture was stirred at 40 ꢁC for 3 h. The resulting mix-
ture was dried under vacuum and the residue purified by
column chromatography using CH2Cl2/CH3OH (30/1
v/v) as eluent. Dimethyl derivative 9 (0.26 g, 30% yield)
was obtained as a yellow solid. Mp 203–204 ꢁC
(DMF/H2O). 1H NMR (CDCl3) d (ppm): 7.58 (d,
3J ¼ 8:8 Hz, 2H), 7.54 (d, 3J ¼ 8:8 Hz, 2H), 6.87 (d,
3J ¼ 8:8 Hz, 2H), 6.84 (d, 3J ¼ 8:8 Hz, 2H), 3.83 (s, 6H),
2.77 (s, 3H), 2.65 (s, 3H). 13C NMR (CDCl3) d (ppm):
175.1, 172.0, 161.5, 161.0, 160.1, 151.8, 150.6, 132.1,
131.8, 130.8, 129.8, 114.2, 114.1, 55.7, 15.3, 13.0. Anal.
Calcd for C22H20N4O2S2: C, 60.55; H, 4.59; N, 12.84; S,
14.68. Found: C, 60.14; H, 4.65; N, 12.55; S, 14.25.
Acknowledgements
Authors thank Dr. Elguero for helpful discussions.
Financial support from CICYT (projet SAF99-98),
UBA (Universidad de Buenos Aires) and CONICET as
well as collaboration from RIIDDMED (Iberoamerican
Network on Investigation and Development of Medi-
cines) of the Iberoamerican Program of Science and
Technology for the Development (CYTED), are grate-
fully acknowledged. Norma B. D’Accorso is member of
CONICET-Argentina.
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