Anticholinesterasic, nematostatic and anthelmintic activities of pyridinic and pyrazinic compounds

Article abstract of DOI:10.2174/092986711796504718

In the search for acetylcholinesterase inhibitors as a potential target for the discovery of anthelmintic drugs, a series of 27 pyridinic and pyrazinic compounds have been designed on the basis of molecular hybridization of two known AChE inhibitors, namely, tacrine and (-)-3-O-acetylspectaline, and on the concept of isosterism. The synthesized compounds generally presented moderate anticholinesterasic activities when compared with the positive control physostigmine, but one compound (ethyl 2-[(6-chloropyrazin-2-yl)sulfanyl] acetate, 11) exhibited an in vitro ability to immobilize the root-knot nematode Meloidogyne incognita that was highly comparable to that of the positive control Temik. Moreover, in anthelmintic assays against the gastrointestinal parasitic nematode Nippostrongylus brasiliensis (L4), some of the compounds, such as (6-chloropyrazin-2-yl)sulfanyl ethanol (32, EC₅₀ = 33 nM), presented activities that were considerably stronger than that of the positive control albendazole (EC₅₀ = 340 nM). In the light of the positive results obtained in the anthelmintic evaluations, the acute oral toxicity of the representative compound diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl] diacetate (7) was determined in rats, and the drug was shown to be non-toxic at a dose of 2000 mg/kg. These results, allied with the relatively simple structures of the active compounds and their facile synthesis, highlight their potential use as anthelmintic or nematicidic agents.

Full text of DOI:10.2174/092986711796504718

Current Medicinal Chemistry, 2011, 18, 3423-3430
3423
Anticholinesterasic, Nematostatic and Anthelmintic Activities of Pyridinic and Pyraz-
inic Compounds
M. Valli1, A. Danuello1, M. Pivatto1, J.C. Saldaña2, H. Heinzen2, L. Domínguez2, V.P. Campos3,
S.R. Marqui1, M.C.M. Young4, C. Viegas Jr.5, D.H.S. Silva1 and V.S. Bolzani*,1
1Departamento de Química, Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), Instituto de Química,
UNESP – Universidade Estadual Paulista, C.P. 355, 14801-970, Araraquara, SP, Brazil
2Facultad de Química, Universidad de la República, General Flores 2124, CC 1157, Montevideo, Uruguay
3Departamento de Fitopatologia,UFLA – Universidade Federal de Lavras, C.P. 3037, CEP 37200-000 Lavras, MG, Brazil
4Seção de Fisiologia e Bioquímica de Plantas, IBt –Instituto de Botânica C.P. 4009, 01061-970, São Paulo, SP, Brazil
5Departamento de Ciências Exatas, Laboratório de Fitoquímica e Química Medicinal (LFQM), Universidade Federal de Alfenas,
37130-000, Alfenas, MG, Brasil
Abstract: In the search for acetylcholinesterase inhibitors as a potential target for the discovery of anthelmintic drugs, a series of 27
pyridinic and pyrazinic compounds have been designed on the basis of molecular hybridization of two known AChE inhibitors, namely,
tacrine and (–)-3-O-acetylspectaline, and on the concept of isosterism. The synthesized compounds generally presented moderate anticho-
linesterasic activities when compared with the positive control physostigmine, but one compound (ethyl 2-[(6-chloropyrazin-2-
yl)sulfanyl] acetate, 11) exhibited an in vitro ability to immobilize the root-knot nematode Meloidogyne incognita that was highly compa-
rable to that of the positive control Temik. Moreover, in anthelmintic assays against the gastrointestinal parasitic nematode Nippostrongy-
lus brasiliensis (L4), some of the compounds, such as (6-chloropyrazin-2-yl)sulfanyl ethanol (32, EC50 = 33 nM), presented activities that
were considerably stronger than that of the positive control albendazole (EC50 = 340 nM). In the light of the positive results obtained in
the anthelmintic evaluations, the acute oral toxicity of the representative compound diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl]
diacetate (7) was determined in rats, and the drug was shown to be non-toxic at a dose of 2000 mg/kg. These results, allied with the rela-
tively simple structures of the active compounds and their facile synthesis, highlight their potential use as anthelmintic or nematicidic
agents.
Keywords: Acetylcholinesterase inhibition, anthelmintic activity, nematicidic activity, pyridine derivatives, molecular hybridization, isoster-
ism, Nippostrongylus brasiliensis, Meloidogyne incognita, natural products.
were evaluated for AChE inhibitory, nematostatic and anthelmintic
activities. In view of the favorable results obtained in the an-
thelmintic assay, the acute oral toxicity of a representative com-
pound, diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl] diace-
tate, was evaluated in rats.
INTRODUCTION
Acetylcholinesterase (AChE) is a serine hydrolase that is ex-
pressed in a wide range of eukaryotes including nematodes and
helminths. The enzyme presents an attractive target in drug discov-
ery, and reversible or irreversible AChE inhibitors have been em-
ployed in numerous applications including the control of parasites.
In this context, nematodes can parasitize humans, animals and
plants, and constitute a serious cause for concern in respect of hu-
man and animal health. In particular, parasitic nematodes represent
a major problem in livestock farming, causing reductions in the
weight and productivity of animals, and elevating their rate of mor-
tality [1]. Additionally, root-knot nematodes have become a signifi-
cant threat in the main coffee-producing countries of the world,
with 17 species of Meloidogyne having been identified as coffee
parasites [2-4].
RESULTS AND DISCUSSION
Synthesis of Potential AChE Inhibitors
In order to synthesize novel compounds with the potential to
control nematodes, two potent AChE inhibitors were selected for
molecular hybridization, namely, the aminoacridine tacrine (1) used
in the treatment of Alzheimer’s disease, and the natural piperidine
alkaloid (–)-3-O-acetylspectaline (2) that has been isolated from
Senna spectabilis [8-10]. A range of isosteric pyridinic and pyraz-
inic compounds were designed, together with a number of hybrid
compounds based on a combination of the pyridine ring of 1 and
the piperidine sub-unit of 2 as outlined in Fig. (1).
The general synthetic route outlined in Scheme 1 was employed
in the preparation of compounds 7 - 33. The commercially available
starting materials 3 - 6 provided the intermediary products 7, 8 and
11 - 15 [11]. The bicyclic compounds 9 and 10 were formed by
reduction of the nitro groups of 7 and 8, respectively [12]. In order
to investigate the contribution to the AChE inhibitory activity and
the parasitic evaluation of the organic substituent on the side chain
of the pyridinic or pyrazinic ring, a series of alcohols, carboxylic
acids and esters were synthesized from the intermediary com-
pounds. Reduction of the ester side chains of 11 and 12 provided
the alcohols 32 and 33, respectively [13]. The carboxylic acids 16 -
21 were obtained by hydrolysis of the ester side chains of com-
pounds 7 - 9 and 11 - 13 [14]. Compounds 16 and 17 were esteri-
fied with MeOH, n-PrOH or n-BuOH under acidic conditions to
yield esters 22 - 27. Reactions with n-PrOH provided yields that
The strategy generally employed to control parasitic nematodes
is based on the use of classic AChE inhibitors such as carbamates
and organophosphates that kill the parasite by impairing movement
or paralyzing the pathogen. However, resistance to the normal che-
motherapeutic agents employed in the control of nematode infec-
tion, and particularly benzimidazoles and last generation iver-
mectins, is well-documented [5-7].
As part of an ongoing research program focused on AChE in-
hibitors, we have synthesized a series of 27 pyridinic and pyrazinic
compounds that were designed on the basis of molecular hybridiza-
tion of two known AChE inhibitors, namely, tacrine and (–)-3-O-
acetylspectaline, and on the concept of isosterism. The compounds
*Address correspondence to this author at the Departamento de Química, Núcleo de
Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), Instituto de
Química, UNESP – Universidade Estadual Paulista, C.P. 355, 14801-970, Araraquara,
SP, Brazil; Tel: +55 16 3301-9660; Fax: +55 16 3301-9692;
E-mail: bolzaniv@iq.unesp.br
0929-8673/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

3424 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
NH2
Valli et al.
N
H
N
O
tacrine (1)
O
CH3
CH3
O
S
N
S
O
New hybrid (9)
O
CH3
H3C
N
H
9
O
(-)-3-O-acetylspectaline (2)
Fig. (1). Typical hybrid product (9) designed by molecular hybridization of tacrine (1) and (–)-3-O-acetylspectaline (2).
N
R1
N
R2
32: R1 = Cl, R2 = SCH2CH2OH
33: R1 = R2 SCH2CH2OH
d), 11 and 12
commercial starting materials
R3
N
R2
R4
R3
N
R2
R3
N
R2
Cl
R3
N
R2
R4
c)
b), 7, 8, 11, 12 and 13
a)
R1
R1
SCH2COOH
R1
R1
22: R1 = R4 = SCH2CO2CH3, R2 = NO2, R3 = CH
23: R1 = R4 = SCH2CO2C3H7, R2 = NO2, R3 = CH
24: R1 = R4 = SCH2CO2C4H9, R2 = NO2, R3 = CH
25: R1 = H, R2 = NO2, R3 = CH, R4 = SCH2CO2CH3
26: R1 = H, R2 = NO2, R3 = CH, R4 = SCH2CO2C3H7
27: R1 = H, R2 = NO2, R3 = CH, R4 = SCH2CO2C4H9
16: R1 = SCH2COOH, R2 = NO2, R3 = CH
17: R1 = H, R2 = NO2, R3 = CH
19: R1 = Cl, R2 = H, R3 = N
20: R1 = SCH2COOH, R2 = H, R3 = N
21: R1 = Cl, R2 = H, R3 = CH
3: R1 = Cl, R2 = NO2, R3 = CH
4: R1 = H, R2 = NO2, R3 = CH
5: R1 = Cl, R2 = H, R3 = N
6: R1 = Cl, R2 = H, R3 = CH
7: R1= R4 = SCH2CO2C2H5, R2 = NO2, R3 = CH
8: R1 = H, R2 = NO2, R3 = CH, R4 = SCH2CO2C2H5
11: R1 = Cl, R2 = H, R3 = N, R4 = SCH2CO2C2H5
12: R1 = R4 = SCH2CO2C2H5, R2 = H, R3 = N
13: R1 = Cl, R2 = H, R3 = CH, R4 = SCH2CO2C2H5
14: R1 = Cl, R2 = NO2, R3 = CH, R4 = SCH2CO2C2H5
15: R1 = SCH2CO2C2H5, R2 = NO2, R3 = CH, R4 = Cl
29: R1 = Cl, R2 = H, R3
= N, R4 = SCH2CO2C3H7
30: R1 = R4 = SCH2CO2C3H7, R2 = H, R3 = N
31: R1 = Cl, R2 = H, R3 = CH, R4 = SCH2CO2C3H7
e), 7 and 8
H
H
H
N
O
N
O
N
O
c)
b), 9
R1
N
S
HOOCH2CS
N
S
C3H7O2CH2CS
N
S
18
28
9: R1 = SCH2CO2C2H5
10: R1 = H
Scheme 1. Synthesis of compounds 7 - 33. Reagents and conditions: a) ethyl-2-mercaptoacetate, NaH, anhydrous THF, N2 atmosphere, room
temperature or ice bath, depending on the reaction, for 5 min to 12 h, depending on the starting material; b) NaOH, EtOH/H2O (2:1), room
temperature for 1 h; c) alcohol (MeOH, n-PrOH or n-BuOH), H2SO4, reflux for 12 h; d) LiAlH4, anhydrous THF, N2 atmosphere, ice bath for
12 h; e) Feo, NH4Cl, EtOH/H2O (2:1), reflux for 90 min.
were higher than those obtainable with MeOH or n-BuOH, hence,
n-PrOH was used in the esterification of 18 - 21 to afford com-
pounds 28 - 31, respectively. To the best of our knowledge, 17 (7,
9, 12, 13, 15, 16, 18, 21, 23, 24, 26 - 31 and 33) of the 27 com-
pounds synthesized in the present study represent new chemical
entities.
limits of detection (LOD) were subsequently determined (Table 1).
Interestingly, the bicyclic compounds 9 and 10, and molecules con-
taining carboxylic acid side chains, i.e. 16 - 21, showed no signifi-
cant AChE inhibitory activities. The LOD values obtained for the
homologous series of side chain esters 7, 22 - 24 and 8, 25 - 27
were in the range 8.6 to 16.0 nmol, and revealed that varying the
number of carbon atoms in the ester moiety from one to four did not
significantly alter the anticholinesterasic activity. The LOD values
of the side chain alcohols 32 and 33 were very high (130 and 100
nmol, respectively) indicating that these compounds were consid-
erably less active than their corresponding esters 11 and 12.
Clearly, the nature of the side chain plays a significant role in
AChE inhibition since activity diminished within the series in the
order esters > alcohols > carboxylic acids. The most active of the
compounds studied were 14 and 15, the LOD values of which were
5.6 and 2.8 nmol, respectively. Such inhibitory activities must be
Biological Evaluations
1. Anticholinesterasic Activity
The inhibitory activities of compounds 7 - 33 against electric
eel AChE were evaluated in vitro using a TLC bioautographic
method [15] with the carbamate physostigmine, a natural AChE
inhibitor, as positive control. An initial screening was performed
using 50 ꢀg of each compound, and for molecules that were consid-
ered to be active, namely, 7, 8, 11 - 15 and 22 - 33, the respective

Controlling Parasitic Nematodes
Current Medicinal Chemistry, 2011 Vol. 18, No. 22
3425
Table 2.
In vitro Nematostatic Activities of the Synthesized
Compounds Presented as Percentage Immobilization of
M. incognita
considered as moderate, however, since LOD values were in the
order of 15- to 35-fold higher than that of physostigmine. However,
since compounds that affect the colinergic system, such as AChE
inhibitors, may have potential value in the control of nematode
parasites, two further evaluations of selected compounds were per-
formed, one using a phytopathogenic nematode (nematostatic activ-
ity) and another using a nematode pathogenic to rats (anthelmintic
activity).
Compound
Nematode immobilization (%)
7
8
12
12
16
19
98
17
18
100
9
Table 1.
In vitro AChE Inhibitory Activities of the Synthesized
Compounds Presented as Limits of Detection (LOD)
10
11
12
13
Compound
AChE inhibitory activity (LOD, nmol)
7
8.6
13.0
13.0
9.8
Positive control (Temik)
8
11
12
13
14
15
22
23
24
25
26
27
28
29
30
31
32
33
with the 50% death rate induced by the positive control. Interest-
ingly, while 11 showed a very high activity against the phytopatho-
genic nematode M. incognita, it was one of the least active com-
pounds in the anthelmintic assay. One explanation for such con-
trasting results is that the nematodes used in the nematostatic and in
the anthelmintic assay were from different species.
13.0
5.6
2.8
9.3
Table 3.
In vitro Anthelmintic Activities of the Synthesized
Com-Compounds Presented as Percentage Death of
N. brasiliensis, (L4)
16.0
15.0
13.0
12.0
10.0
42.0
50.0
70.0
25.0
130.0
100.0
0.2
Compound
Concentration in well (ꢀM)
Nematode death (%)
7
0.138
0.210
0.176
0.301
0.215
0.158
0.216
0.181
0.181
0.164
0.234
0.195
0.245
0.192
0.246
0.150
0.128
0.120
0.219
0.195
0.185
0.168
0.260
0.215
56.5
49.5
60.2
56.2
42.0
46.7
55.8
57.4
55.5
50.8
54.0
71.8
58.0
56.0
72.7
59.0
62.5
49.6
56.7
70.3
42.1
64.0
74.0
63.4
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
32
33
Positive control (physostigmine)
2. Nematostatic Activity
Compounds 7 - 13 were evaluated for their ability to immobi-
lize second-stage juveniles of the root-knot nematode Meloidogyne
incognita in an in vitro assay. Each compound was tested at a con-
centration of 500 ppm alongside the carbamate pesticide Temik (the
active component of which is aldicarb) as positive control, and the
percentage immobilization of the nematode was determined after 24
h exposure. As shown in Table 2, compound 11 was the most po-
tent of the series with 98% of nematodes immobilized. A compari-
son of the structures and activities of the assayed compounds re-
vealed that the chloro substituent at C-6 and the pyrazine ring were
important for activity, while compounds with a nitro substituent at
C-3 were less active. The high activities of the tested compounds,
and especially that of 11, in comparison with the activity of the
positive control highlights the potential use of these compounds for
parasite management.
Positive control
(albendazole)
0.340
50.0
3. Anthelmintic Activity
The EC50 values of selected compounds that exhibited a rea-
sonably high death rate in the initial screening and were of rela-
tively simple structure were subsequently determined against N.
brasiliensis (L4) (Table 4). However, the anthelmintic properties of
the compounds studied were somewhat similar and, hence, it was
not possible to draw reliable structure/activity relationships for
these compounds. All compounds were considered to possess rea-
sonably high anthelmintic activities, while compounds 10 and 32
showed EC50 values (34.0 and 33.0 nM, respectively) that were 10-
fold lower than that of albendazole (EC50 = 340 nM), hence indicat-
The anthelmintic activities of compounds 7 - 28 and 32 - 33
were evaluated against L4 larvae of Nippostrongylus brasiliensis, a
gastrointestinal parasitic nematode of rats. The screening was ini-
tially performed using a fixed concentration of test compound (0.05
ꢀg/mL) that was in the same order of magnitude as the EC50 value
of the positive control albendazole, and the percentage death rates
of nematode larvae were recorded after a 5-day exposure. As shown
in Table 3, application of the test compounds at 0.05 ꢀg/mL re-
sulted in the death of 42 to 74% of the nematodes in comparison

3426 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
Valli et al.
ing significant activity. It is important to stress, however, that the
mechanisms associated with the activities observed in the nema-
tostatic and anthelmintic assays have not been established, and such
activities may not be related to AChE inhibition. Further chemical
and pharmacological investigations would be required in order to
establish the possible mechanisms of action of the studied com-
pounds.
useful in the control of parasites does allow the discovery of novel
compounds with potent anthelmintic activity.
EXPERIMENTAL
General
Reagents were purchased from Aldrich, Acros Organics or
Fluka and were used without further treatment; solvents were puri-
fied according to standard methods [17]. Reaction progress was
monitored by TLC on glass plates pre-coated with silica gel F254
layers (0.2 mm thickness; Whatman) and visualized under UV light
(254 and 366 nm) and after spraying with anisaldehyde–H2SO4
followed by charring for 5 min. Preparative TLC was performed on
glass plates pre-coated with silica gel F254 (particle size 5 - 50 ꢀm;
Macherey-Nagel) and visualized under UV light at 254 nm. Col-
umn chromatography (CC) was carried out over silica gel (particle
size 35 - 70 ꢀm; Acros Organics). Melting points were measured on
a Microquímica model MQAPF-302 apparatus. IR spectra were
recorded on a Jasco model 4100 FT/IR spectrometer in the attenu-
ated total reflectance (ATR) mode. NMR spectra were recorded at
30 °C on a Varian Inova 500 (11.7 T) instrument operating at 500
MHz (1H) and 125 MHz (13C); spectra were referenced against the
internal standard TMS (ꢀTMS = 0.00) or against the residual solvent
resonances of CDCl3 (ꢀ = 7.26 [1H] and 77.0 [13C] ppm) or DMSO-
d6 (ꢀ = 2.50 [1H] and 39.5 [13C] ppm). Some 13C values were attrib-
uted using evidence from gHMBC and are marked with an asterisk
in the data provided. Electrospray ionization – time-of-flight (ESI-
TOF) high resolution mass spectra were measured on a Bruker-
Daltonics model ultrOTOFQ spectrometer operating in the positive
ion mode. ChemDraw Ultra 8.0 was used to calculate the values of
the pseudomolecular ions [M+H]+ and [M+Na]+.
Table 4.
EC50 Values for Tested Compounds that Presented the
Most Effective Anthelmintic Activities in the Initial Screen-
ing
Compound
EC50 (nM)
7
56.0
34.0
10
17
230.0
60.0
21
26
69.0
32
33.0
Positive control (albendazole)
340.0
4. Acute Toxicity Assay
In the light of the encouraging results obtained in the nema-
tostatic and anthelmintic evaluations, an acute oral toxicity test was
performed in rats. Compound 7 was selected for this evaluation
because it presented a very high yield in synthesis and showed high
activity in the anthelmintic evaluation. The assay was carried out in
accordance with the Organization for Economic Cooperation and
Development (OECD 425) guidelines [16] and involved oral ad-
ministration of 2000 mg/kg of 7 to each of five female Wistar rats.
The rats were observed over 14 days, during which time no signifi-
cant variations in behavior occurred and recorded increases in the
body weights of the animals were within normal limits. The tested
dose did not induce death in any of the experimental animals. The
animals were sacrificed at the end of the observational period and
submitted to necropsy. No abnormalities were registered in any of
the experimental animals. It is concluded, therefore, the LD50 value
of 7 is greater than 2000 mg/kg and that the compound must be
considered non-toxic at this dose level. This result is particularly
favorable given the high anthelmintic activities of the studied series
of compounds.
Synthetic methods
1. Diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl] diacetate
(7)
Ethyl-2-mercaptoacetate (716 ꢁL, 6.5 mmol) was added with
constant stirring to NaH (156 mg, 6.5 mmol) dissolved in anhy-
drous THF (50 mL) and maintained under a nitrogen atmosphere.
After 30 min, 2,6-dichloro-3-nitropyridine (3) (500 mg, 2.6 mmol)
was added to the solution and stirring continued for 15 min. At
completion of reaction (monitored by TLC) cold water was poured
onto the reaction mixture and the whole extracted with EtOAc. The
organic layer was dried over MgSO4, the solvent was evaporated
under reduced pressure, and the resulting crude product subjected to
CC eluted with n-hexane:EtOAc (gradient from 7:3 to 0:1) to afford
7 (902 mg, 97% yield) as a pure yellow solid [11]: TLC Rf 0.35 (n-
CONCLUSIONS
The 27 pyridinic and pyrazinic compounds synthesized in the
present study presented moderate anticholinesterasic activity in
comparison with the positive control physostigmine. In contrast,
ethyl 2-[(6-chloropyrazin-2-yl)sulfanyl] acetate (11) was very po-
tent in the screening assay against the phytopathogen Meloidogyne
incognita, and induced immobilization in 98% of nematodes, a
result that was highly comparable with that of the commercial pes-
ticide Temik. In assays against the gastrointestinal parasitic nema-
tode Nippostrongylus brasiliensis, all compounds exhibited an-
thelmintic activity while some, for example (6-chloropyrazin-2-
yl)sulfanyl ethanol (32; EC50 = 33 nM), showed activities that were
considerably higher than that of the positive control albendazole
(EC50 = 340 nM). The acute oral toxicity of a representative com-
pound, namely, diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissul-
fanediyl] diacetate (7), was evaluated in rats and the drug was dem-
onstrated to be non-toxic at a dose of 2000 mg/kg. These results
highlight the potential use of this class of compound as anthelmintic
or nematicidic agents. Additionally, the compounds have relatively
simple structures and their syntheses may be accomplished readily
even on a large-scale. The results presented herein demonstrate that
application of the rational approach that AChE inhibitors can be
o
hexane–EtOAc, 7:3); MP 81 - 82 C; IR (ATR) ꢀ cm–1: 2977, 2373,
1741, 1550; 1H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.19 (m, 6H), 3.92
(s, 2H), 4.01 (s, 2H), 4.14 (m, 4H), 7.01 (d, 1H, J = 8.5 Hz), 8.24
(d, 1H, J = 8.5 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 14.0,
32.3, 33.1, 61.8, 62.1, 117.1, 133.2, 139.0*, 157.0, 163.8, 168.4,
168.9; HRMS (ESI–TOF) m/z: 361.0579 [M+H]+ (calcd for
C13H17N2O6S2, 361.0523), 383.0478 [M+Na]+ (calcd for
C13H16N2O6S2Na, 383.0342).
2. Ethyl 2-[(3-nitropyridin-2-yl)sulfanyl] acetate (8)
Ethyl-2-mercaptoacetate (716 ꢁL, 6.5 mmol) was added with
constant stirring to NaH (160 mg, 6.6 mmol) dissolved in anhy-
drous THF (50 mL) and maintained under a nitrogen atmosphere.
After 30 min, 2-chloro-3-nitropyridine (4) (700 mg, 4.4 mmol) was
added to the solution and stirring continued for 30 min. At comple-
tion of reaction (monitored by TLC) cold water was poured onto the
reaction mixture and the whole extracted with EtOAc. The organic
layer was dried over MgSO4, the solvent was evaporated under
reduced pressure, and the resulting crude product subjected to CC
eluted with n-hexane:EtOAc (gradient from 7:3 to 0:1) to afford 8
(1020 mg, 96% yield) as a pure orange solid [11]: TLC Rf 0.40 (n-

Controlling Parasitic Nematodes
Current Medicinal Chemistry, 2011 Vol. 18, No. 22
3427
o
1
hexane–EtOAc, 7.3); MP 55 - 56 C; IR (ATR)ꢀ cm–1:3082, 2982,
(n-hexane–EtOAc, 7:3); H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.21
(t, 6H, J = 7.0 Hz), 3.89 (s, 4H), 4.16 (q, 4H, J = 7.0 Hz), 8.12 (s,
2H); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 14.0, 31.7, 61.8, 138.2,
154.1, 168.8; HRMS (ESI–TOF) m/z: 317.0629 [M+H]+ (calcd for
C12H17N2O4S2, 317.0624).
1
1731, 1583; H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.19 (t, 3H, J =
7.0 Hz), 3.88 (s, 2H), 4.14 (q, 2H, J = 7.0 Hz), 7.16 (dd, 1H, J = 5.0
and 8.5 Hz), 8.44 (dd, 1H, J = 1.5 and 8.5 Hz), 8.57 (dd, 1H, J = 1.5
and 5.0 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 14.2, 33.4, 61.6,
119.2, 133.7, 141.0*, 152.9, 156.4, 169.1; HRMS (ESI–TOF) m/z:
243.0422 [M+H]+ (calcd for C9H11N2O4S, 243.0434), 265.0235
[M+Na]+ (calcd for C9H10N2O4SNa, 265.0253).
6. Ethyl 2-[(6-chloropyridin-2-yl)sulfanyl] acetate (13)
Ethyl-2-mercaptoacetate (1560 ꢂL, 14 mmol) was added with
constant stirring to NaH (340 mg, 14 mmol) dissolved in anhydrous
THF (50 mL) and maintained under a nitrogen atmosphere and
cooled in an ice bath. After 30 min, 2,6-dichloro-pyridine (6) (700
mg, 4.7 mmol) was added to the solution and stirring continued for
12 h. At completion of reaction (monitored by TLC) cold water was
poured onto the reaction mixture and the whole extracted with
EtOAc. The organic layer was dried over MgSO4, the solvent was
evaporated under reduced pressure, and the resulting crude product
subjected to preparative TLC and developed twice with n-
hexane:EtOAc (9:1) to afford 13 (90.5 mg, 29% yield) as a pure
3. Ethyl 2-[(2,3-dihydro-2-oxo-1H-pyrido[2,3-b][1,4]thiazin-6-
yl)sulfanyl] acetate (9)
Iron powder (350 mg, 6.2 mmol) and ammonium chloride (35
mg, 0.7 mmol) were added with constant stirring to a solution con-
taining 250 mg (1.03 mmol) of diethyl 2,2'-[(3-nitropyridine-2,6-
diyl) bissulfanediyl] diacetate (7) in 15 mL EtOH/H2O (2:1). The
reaction mixture was heated under reflux until complete consump-
tion of the reagents (90 min; monitored by TLC), cooled to room
temperature, filtered and extracted with EtOAc. The organic layer
was dried over MgSO4, the solvent was evaporated under reduced
pressure, and the resulting crude product subjected to CC eluted
with n-hexane:EtOAc (gradient from 7:3 to 0:1) to afford 9 (128
mg, 65% yield) as a pure light brown solid [12]: TLC Rf 0.62 (n-
hexane–EtOAc, 1:9); MP 151 - 152 oC; IR (ATR)ꢀ cm–1: 3082,
1
colorless oil [11]: TLC Rf 0.64 (n-hexane–EtOAc, 7:3); H-NMR
(500 MHz, CDCl3) ꢀ ppm: 1.22 (t, 3H, J = 7.0 Hz), 3.86 (s, 2H),
4.16 (q, 2H, J = 7.0 Hz), 6.94 (dd, 1H, J = 0.5 and 8.0 Hz), 7.07
(dd, 1H, J = 0.5 and 8.0 Hz), 7.37 (dd, 1H, J = 8.0 Hz); 13C-NMR
(125 MHz, CDCl3) ꢀ ppm: 14.1, 32.7, 61.7, 119.9, 120.2, 138.4,
151.0, 158.0, 169.2; HRMS (ESI–TOF) m/z: 232.0192 [M+H]+
(calcd for C9H11ClNO2S, 232.0194), 254.0006 [M+Na]+ (calcd for
C9H10ClNO2SNa, 254.0013).
1
2982, 1722, 1659; H-NMR (500 MHz, DMSO-d6) ꢀ ppm: 1.23 (t,
3H, J = 7.0 Hz), 3.46 (s, 2H), 3.83 (s, 2H), 4.16 (q, 2H, J = 7.0 Hz),
6.89 (d, 1H, J = 8.5 Hz), 6.92 (d, 1H, J = 8.5 Hz), 8.6 (br s, 1H);
13C-NMR (125 MHz, DMSO-d6) ꢀ ppm: 14.2, 29.7, 33.0, 61.7,
119.6, 124.7, 129.7, 143.5, 151.3, 165.0, 169.7; HRMS (ESI–TOF)
m/z: 285.0390 [M+H]+ (calcd for C11H13N2O3S2, 285.0362),
307.0249 [M+Na]+ (calcd for C11H12N2O3S2Na, 307.0182).
7. Ethyl 2-[(6-chloro-3-nitropyridin-2-yl)sulfanyl] acetate (14)
and ethyl 2-(6-chloro-5-nitropyridin-2-yl)sulfanyl] acetate (15)
Ethyl-2-mercaptoacetate (160 ꢂL, 1.45 mmol) was added with
constant stirring to NaH (35 mg, 1.45 mmol) dissolved in anhy-
drous THF (50 mL) and maintained under a nitrogen atmosphere
and cooled in an ice bath. After 30 min, 2,6-dichloro-3-
nitropyridine (3) (400 mg, 2.07 mmol) was added to the solution
and stirring continued for 5 min, following which cold water was
poured onto the reaction mixture and the whole extracted with
EtOAc. The organic layer was dried over MgSO4, the solvent was
evaporated under reduced pressure, and the resulting crude product
subjected to preparative TLC and developed three times with n-
hexane:EtOAc (9:1) to afford 14 (128.8 mg, 32% yield) and 15 (64
mg, 16% yield) as pure yellow solids [11]. Compound 14: TLC Rf
4. 1H-pyrido[2,3-b][1,4]thiazin-2(3H)-one (10)
Iron powder (700 mg, 12.5 mmol) and ammonium chloride (70
mg, 1.3 mmol) were added with constant stirring to a solution con-
taining 500 mg (2.06 mmol) of ethyl 2-[(3-nitropyridin-2-
yl)sulfanyl] acetate (8) in 15 mL EtOH/H2O (2:1). The reaction
mixture was heated under reflux for 90 min (monitored by TLC),
cooled to room temperature, filtered and extracted with EtOAc. The
organic layer was dried over MgSO4, the solvent was evaporated
under reduced pressure, and the resulting crude product subjected to
CC eluted with n-hexane:EtOAc (gradient from 7:3 to 0:1) to afford
10 (52 mg, 15% yield) as a pure light brown solid [12]: TLC Rf 0.28
0.53 (n-hexane–EtOAc, 7.3); MP 58.7 - 59.8 C; IR (ATR)ꢀ cm–
o
(n-hexane–EtOAc, 3:7); MP 198 - 199 C; IR (ATR)ꢀ cm–1:3121,
1:2920, 1726, 1568; H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.23 (t,
o
1
1
1673; H-NMR (500 MHz, DMSO-d6) ꢀ ppm: 3.64 (s, 2H), 7.18
3H, J = 7.0 Hz), 3.84 (s, 2H), 4.17 (q, 2H, J = 7.0 Hz), 7.13 (d, 1H,
J = 8.5 Hz), 8.39 (d, 1H, J = 8.5 Hz); 13C-NMR (125 MHz, CDCl3)
ꢀ ppm: 14.2, 34.0, 61.8, 119.7, 136.0, 140.0*, 154.7, 157.9, 168.6;
HRMS (ESI–TOF) m/z: 277.0049 [M+H]+ (calcd for
C9H10ClN2O4S, 277.0044). Compound 15: TLC Rf 0.44 (n-hexane–
EtOAc, 7:3); MP 77.0 - 78.0 oC. IR (ATR)ꢀ cm–1:3082, 2997,
(dd, 1H, J = 5.0 and 8.0 Hz), 7.26 (dd, 1H, J = 1.5 and 8.0 Hz), 8.08
(dd, 1H, J = 1.5 and 5.0 Hz), 10.59 (br s, 1H); 13C-NMR (125 MHz,
DMSO-d6) ꢀ ppm: 28.7, 121.5, 123.5, 133.7, 143.0*, 143.1, 164.5;
HRMS (ESI–TOF) m/z: 167.0275 [M+H]+ (calcd for C7H7N2OS,
167.0274).
1
1741, 1559; H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.23 (t, 3H, J =
5. Ethyl 2-[(6-chloropyrazin-2-yl)sulfanyl] acetate (11) and diethyl
2,2'-[(pyrazin-2,6-diyl) bissulfanediyl] diacetate (12)
7.0 Hz), 3.92 (s, 2H), 4.16 (q, 2H, J = 7.0 Hz), 7.22 (d, 1H, J = 8.5
Hz), 8.04 (d, 1H, J = 8.5 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm:
14.1, 33.1, 62.1, 120.3, 134.2, 141.0*, 143.8, 163.5, 168.2; HRMS
(ESI–TOF) m/z: 277.0049 [M+H]+ (calcd for C9H10ClN2O4S,
277.0044), 298.9869 [M+Na]+ (calcd for C9H9ClN2O4SNa,
298.9864).
Ethyl-2-mercaptoacetate (880 ꢂL, 8 mmol) was added with
constant stirring to NaH (960 mg, 8 mmol) dissolved in anhydrous
THF (50 mL) and maintained under a nitrogen atmosphere and
cooled in an ice bath. After 30 min, 2,6-dichloro-pyrazine (5) (400
mg, 2.6 mmol) was added to the solution and stirring continued for
2 h. At completion of reaction (monitored by TLC) cold water was
poured onto the reaction mixture and the whole extracted with
EtOAc. The organic layer was dried over MgSO4, the solvent was
evaporated under reduced pressure, and the resulting crude product
subjected to CC eluted with n-hexane:EtOAc (9:1) to afford 11
(252.5 mg, 42% yield) and 12 (270 mg, 32% yield) as pure color-
less oils [11]. Compound 11: TLC Rf 0.33 (n-hexane–EtOAc, 7:3);
1H-NMR (500 MHz, CDCl3) ꢀ ppm: 1.21 (t, 3H, J = 7.0 Hz), 3.86
(s, 2H), 4.14 (q, 2H, J = 7.0 Hz), 8.15 (s, 1H), 8.32 (s, 1H); 13C-
NMR (125 MHz, CDCl3) ꢀ ppm: 14.0, 32.1, 61.8, 139.0, 140.8,
148.4, 155.0, 168.3; HRMS (ESI–TOF) m/z: 233.0147 [M+H]+
(calcd for C8H10ClN2O2S, 233.0146). Compound 12: TLC Rf 0.10
8. General Method of Synthesis of Compounds 16 - 21
A solution containing NaOH (28 mg, 0.7 mmol) in distilled wa-
ter (5 mL) was added to 7 (100 mg, 0.27 mmol) dissolved in EtOH
(10 mL). The reaction mixture was stirred for 1 h at room tempera-
ture and monitored by TLC, following which the solvent was
evaporated, water was poured onto the concentrate and the resulting
solution neutralized with 1 M HCl to afford compound 16 [14].
Compounds 8, 9 and 11 - 13 were treated in a similar fashion to
afford products 17 - 21, respectively. Compound 16 (2,2'-[(3-
nitropyridin-2,6-diyl)bissulfanediyl] diacetic acid) was obtained as
a yellow solid in 97% yield: TLC Rf 0.28 (EtOAc–MeOH, 1:1); MP
– decomposed at 170 oC before melting; IR (ATR)ꢀ cm–1:3082,

3428 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
Valli et al.
1
1712, 1559; H-NMR (500 MHz, DMSO-d6) ꢀ ppm: 3.76 (s, 2H),
(dipropyl 2,2'-[(3-nitropyridine-2,6-diyl)disulfanediyl] diacetate) was
3.82 (s, 2H), 7.15 (d, 1H, J = 8.5 Hz), 8.26 (d, 1H, J = 8.5 Hz); 13C-
NMR (125 MHz, DMSO-d6) ꢀ ppm: 36.2*, 36.6*, 116.2, 132.8,
137.2, 158.8, 166.8, 169.9*, 170.5*; HRMS (ESI–TOF) m/z:
326.9721 [M+Na]+ (calcd for C9H8N2O6S2Na, 326.9716). Com-
pound 17 (2-[(3-nitropyridin-2-yl)sulfanyl] acetic acid) was ob-
tained as a yellow solid in 96% yield: TLC Rf 0.48 (EtOAc–MeOH,
obtained as a yellow solid in 89% yield: TLC Rf 0.44 (n-hexane–
o
EtOAc, 1:1); MP 50 - 51 C; IR (ATR) ꢀ cm–1:2968, 1731, 1559;
1H-NMR (500 MHz, CDCl3) ꢀ ppm: 0.91 (m, 6H), 1.66 (m, 4H),
4.01 (s, 2H), 4.10 (s, 2H), 4.11 (m, 4H), 7.09 (d, 1H, J = 9.0 Hz),
8.31 (d, 1H, J = 9.0 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 10.2,
21.8, 32.3, 33.1, 67.4, 67.6, 117.1, 133.3, 139.0*, 157.0, 163.8,
168.5, 169.0; HRMS (ESI–TOF) m/z: 389.0841 [M+H]+ (calcd for
C15H21N2O6S2, 389.0836), 411.0676 [M+Na]+ (calcd for
C15H20N2O6S2Na, 411.0655). Compound 24 (dibutyl 2,2'-[(3-
nitropyridine-2,6-diyl)disulfanediyl] diacetate) was obtained as a
1:1); MP 98 - 99 C; IR (ATR)ꢀ cm–1:3288, 1607, 1511; H-NMR
(500 MHz, DMSO-d6) ꢀ ppm: 3.76 (s, 2H), 7.34 (dd, 1H, J = 4.5
and 8.5 Hz), 8.52 (dd, 1H, J = 1.5 and 8.5 Hz), 8.73 (dd, 1H J = 1.5
and 4.5 Hz); 13C-NMR (125 MHz, DMSO-d6) ꢀ ppm: 34.9, 119.6,
134.1, 141.6, 153.6, 156.5, 170.0; HRMS (ESI-TOF) m/z: 215.0119
[M+H]+ (calcd for C7H7N2O4S, 215.0121). Compound 18 ([(2-oxo-
2,3-dihydro-1H-pyrido[2,3-b][1,4]thiazin-6-yl)sulfanyl] acetic acid)
was obtained as a rose-colored solid in 95% yield: TLC Rf 0.44
o
1
1
yellow oil in 24% yield: TLC Rf 0.62 (n-hexane–EtOAc, 1:1); H-
NMR (500 MHz, CDCl3) ꢀ ppm: 0.80 (m, 6H), 1.22 (m, 4H), 1.50
(m, 4H), 3.90 (s, 2H), 3.99 (s, 2H), 4.05 (m, 4H), 7.00 (d, 1H, J =
8.5 Hz), 8.22 (d, 1H, J = 9.0 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ
ppm: 13.6, 19.0, 30.5, 32.3, 33.1, 65.6, 65.9, 117.1, 133.3, 139.0*,
157.0, 163.8, 168.5, 169.0; HRMS (ESI–TOF) m/z: 417.1154
[M+H]+ (calcd for C17H25N2O6S2, 417.1149). Compound 25
(methyl [3-nitropyridin-2-yl)sulfanyl] acetate) was obtained as an
orange solid in 18% yield: TLC Rf 0.62 (n-hexane–EtOAc, 1:1);
o
(EtOAc–MeOH, 1:1); MP – decomposed at 230 C before melting;
IR (ATR)ꢀ cm–1:3174, 1650, 1573; H-NMR (500 MHz, DMSO-
1
d6) ꢀ ppm: 3.57 (s, 2H), 3.59 (s, 2H), 7.04 (d, 1H, J = 8.5 Hz), 7.14
(d, 1H, J = 8.5 Hz), 10.55 (s, 1H); 13C-NMR (125 MHz, DMSO-d6)
ꢀ ppm: 28.8, 37.2, 118.8, 124.6, 129.9, 142.2, 153.7, 164.0, 170.2;
HRMS (ESI–TOF) m/z: 257.0058 [M+H]+ (calcd for C9H9N2O3S2,
257.0049). Compound 19 ([(6-chloropyrazin-2-yl)sulfanyl] acetic
acid) was obtained as a rose-colored solid in 93% yield; TLC Rf
0.44 (EtOAc–MeOH, 1:1). MP – decomposed at 250 oC before
melting; IR (ATR)ꢀ cm–1:3388, 1588; 1H-NMR (500 MHz,
DMSO-d6) ꢀ ppm: 3.72 (s, 2H), 8.34 (s, 1H), 8.51 (s, 1H); 13C-
NMR (125 MHz, DMSO-d6) ꢀ ppm: 36.1, 137.7, 140.9, 147.0,
158.9, 169.1; HRMS (ESI–TOF) m/z: 204.9833 [M+H]+ (calcd for
C6H6ClN2O2S, 204.9833). Compound 20 (2,2'-[(pyrazin-2,6-
diyl)bissulfanediyl] diacetic acid) was obtained as a rose-colored
solid in 92% yield; TLC Rf 0.37 (EtOAc–MeOH, 1:1); MP – de-
o
MP 53 - 54 C; IR (ATR)ꢀ cm–1: 2954, 1722, 1555; 1H-NMR (500
MHz, CDCl3) ꢀ ppm: 3.68 (s, 2H), 3.92 (s, 2H), 7.16 (dd, 1H, J =
4.5 and 8.0 Hz), 8.45 (dd, 1H, J = 2.0 and 8.0 Hz), 8.59 (dd, 1H, J =
2.0 and 4.5 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 33.1, 52.6,
119.3, 133.7, 142.0*, 152.9, 156.3, 169.6; HRMS (ESI–TOF) m/z:
229.0283 [M+H]+ (calcd for C8H9N2O4S, 229.0278), 251.0097
[M+Na]+ (calcd for C8H8N2O4SNa, 251.0097). Compound 26 (pro-
pyl [3-nitroropyridin-2-yl)sulfanyl] acetate) was obtained as an
1
orange oil in 69% yield: TLC Rf 0.66 (n-hexane–EtOAc, 1:1); H-
NMR (500 MHz, CDCl3) ꢀ ppm: 0.84 (t, 3H, J = 7.0 Hz), 1.58 (m,
2H), 3.90 (s, 2H), 4.04 (t, 2H, J = 7.0 Hz), 7.16 (dd, 1H, J = 4.5 and
8.5 Hz), 8.44 (dd, 1H, J = 1.5 and 8.5 Hz), 8.57 (dd, 1H, J = 1.5 and
4.5 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 10.3, 21.9, 33.4,
67.1, 119.2, 133.7, 142.0*, 152.8, 156.5, 169.2; HRMS (ESI–TOF)
m/z: 257.0596 [M+H]+ (calcd for C10H13N2O4S, 257.0591),
279.0397 [M+Na]+ (calcd for C10H12N2O4SNa, 279.0410). Com-
pound 27 (butyl [3-nitroropyridin-2-yl)sulfanyl] acetate) was ob-
tained as a yellow oil in 59% yield: TLC Rf 0.66 (n-hexane–EtOAc,
composed at 300 C before melting; IR (ATR)ꢀ cm–1: 3226, 1583;
o
1H-NMR (500 MHz, DMSO-d6) ꢀ ppm: 3.63 (s, 4H), 8.04 (s, 2H);
13C-NMR (125 MHz, DMSO-d6) ꢀ ppm: 36.4, 136.1, 157.1, 169.6;
HRMS (ESI–TOF) m/z: 261.0000 [M+H]+ (calcd for C8H9N2O4S2,
260.9998). Compound 21 (2-(6-chloropyridin-2-yl)sulfanyl] acetic
acid) was obtained as a light-brown solid in 96% yield: TLC Rf 0.42
o
(EtOAc–MeOH, 1:1): MP – decomposed at 250 C before melting;
1
1H-NMR (500 MHz, DMSO-d6) ꢀ ppm: 3.58 (s, 2H), 7.09 (dd, 1H,
J = 8.0 and 0.5 Hz), 7.22 (dd, 1H, J = 8.0 and 0.5 Hz), 7.61 (dd, 1H,
J = 8.0 Hz); 13C-NMR (125 MHz, DMSO-d6) ꢀ ppm: 37.3, 118.6,
119.8, 139.4, 149.2, 162.6, 169.7; HRMS (ESI–TOF) m/z:
203.9886 [M+H]+ (calcd for C7H7ClNO2S, 203.9881).
1:1); H-NMR (500 MHz, CDCl3) ꢀ ppm: 0.83 (t, 3H, J = 7.0 Hz),
1.27 (m, 2H), 1.54 (m, 2H), 3.89 (s, 2H), 4.07 (t, 2H, J = 7.0 Hz),
7.16 (dd, 1H, J = 4.5 and 8.5 Hz), 8.44 (dd, 1H, J = 1.5 and 8.5 Hz),
8.57 (dd, 1H, J = 1.5 and 4.5 Hz); 13C-NMR (125 MHz, CDCl3) ꢀ
ppm: 13.6, 19.0, 30.6, 33.4, 65.4, 119.2, 133.7, 142.0*, 152.8,
156.5, 169.2; HRMS (ESI–TOF) m/z: 271.0752 [M+H]+ (calcd for
C11H15N2O4S, 271.0747), 293.0508 [M+Na]+ (calcd for
C11H14N2O4SNa, 293.0566). Compound 28 (propyl [2-oxo-2,3-
dihydro-1H-pyrido[2,3-b][1,4]thiazin-6-yl-sulfanyl] acetate) was
obtained as a yellow solid in 65% yield: TLC Rf 0.33 (n-hexane–
9. General Method of Synthesis of Compounds 22 - 31
An aliquot (1 mL) of H2SO4 was added to 16 (120 mg, 0.4
mmol) dissolved in MeOH (50 mL) and the reaction mixture heated
under reflux for 12 h and monitored by TLC. Following completion
of reaction, the solvent was evaporated, water was poured onto the
concentrate and the resulting solution neutralized with 1 M NaOH
and extracted with EtOAc. The organic layer was dried over
MgSO4, the solvent was evaporated under reduced pressure, and the
resulting crude product subjected to CC eluted with n-
hexane:EtOAc (gradient from 7:3 to 0:1) to afford 22. Product 25
was prepared from 17 using the method described above, products
24 and 27 were prepared from 16 and 17, respectively, in the pres-
ence of n-BuOH as reagent and solvent, and products 23, 26 and 28
- 31 were prepared from 16 - 21, respectively, in the presence of n-
PrOH as reagent and solvent. Compound 22 (dimethyl 2,2'-[(3-
nitropyridine-2,6-diyl)disulfanediyl] diacetate) was obtained as a
yellow solid in 45% yield: TLC Rf 0.44 (n-hexane–EtOAc, 1:1);
1
EtOAc, 1:1); IR (ATR)ꢀ cm–1:3082, 2935, 1736, 1678; H-NMR
(500 MHz, CDCl3) ꢀ ppm: 0.88 (t, 3H, J = 7.0 Hz), 1.62 (m, 2H),
3.46 (s, 2H), 3.85 (s, 2H), 4.06 (t, 2H, J = 7.0 Hz), 6.91 (d, 1H, J =
8.5 Hz), 6.93 (d, 1H, J = 8.5 Hz), 9.25 (s, 1H); 13C-NMR (125
MHz, CDCl3) ꢀ ppm: 10.3, 21.9, 29.7, 32.9, 67.3, 119.6, 124.7,
129.7, 143.5, 151.3, 165.0, 169.8; HRMS (ESI–TOF) m/z:
299.0524 [M+H]+ (calcd for C12H15N2O3S2, 299.0519). Compound
29 (propyl [6-chloropyrazin-2-yl)sulfanyl] acetate) was obtained as
a yellow oil in 41% yield: TLC Rf 0.77 (n-hexane–EtOAc, 1:1); 1H-
NMR (500 MHz, CDCl3) ꢀ ppm: 0.86 (t, 3H, J = 7.0 Hz), 1.61 (m,
2H), 3.88 (s, 2H), 4.07 (t, 2H, J = 7.0 Hz), 8.17 (s, 1H), 8.34 (s,
1H); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 10.3, 21.9, 32.2, 67.5,
139.1, 140.9, 149.0*, 155.0*, 168.5; HRMS (ESI–TOF) m/z:
247.0310 [M+H]+ (calcd for C9H12ClN2O2S, 247.0303). Compound
30 (dipropyl 2,2'-[(pyrazin-2,6-diyl)bissulfanediyl] diacetate) was
obtained as a yellow oil in 51% yield: TLC Rf 0.66 (n-hexane–
o
1
MP 86 - 87 C; IR (ATR)ꢀ cm–1:2964, 1746, 1555; H-NMR (500
MHz, CDCl3) ꢀ ppm: 3.68 (s, 3H), 3.70 (s, 3H), 3.92 (s, 2H), 4.02
(s, 2H), 7.02 (d, 1H, J = 8.5 Hz), 8.24 (d, 1H, J = 8.5 Hz); 13C-
NMR (125 MHz, CDCl3) ꢀ ppm: 32.0, 32.8, 52.7, 52.9, 117.2,
133.3, 139.0*, 157.0, 163.7, 169.0, 169.4; HRMS (ESI–TOF) m/z:
333.0215 [M+H]+ (calcd for C11H13N2O6S2, 333.0210), 355.0044
[M+Na]+ (calcd for C11H12N2O6S2Na, 355.0029). Compound 23
1
EtOAc, 1:1); H-NMR (500 MHz, CDCl3) ꢀ ppm: 0.91 (t, 6H, J =
7.0 Hz), 1.64 (m, 4H), 3.96 (s, 4H), 4.10 (t, 4H, J = 7.0 Hz), 8.19 (s,
2H); 13C-NMR (125 MHz, CDCl3) ꢀ ppm: 10.2, 21.9, 31.7, 67.5,
138.2, 154.2, 168.9; HRMS (ESI–TOF) m/z: 345.0943 [M+H]+
(calcd for C14H21N2O4S2, 345.0937). Compound 31 (propyl [6-

Controlling Parasitic Nematodes
Current Medicinal Chemistry, 2011 Vol. 18, No. 22
3429
chloropyridin-2-yl)sulfanyl] acetate) was obtained as a yellow oil in
20% yield; TLC Rf 0.57 (n-hexane–EtOAc, 1:1); 1H-NMR (500
MHz, CDCl3) ꢀ ppm: 0.86 (t, 3H, J = 7.0 Hz), 1.60 (m, 2H), 3.88 (s,
2H), 4.07 (t, 2H, J = 7.0 Hz), 6.94 (dd, 1H, J = 0.5 and 8.0 Hz),
7.07 (dd, 1H, J = 0.5 and 8.0 Hz), 7.36 (dd, 1H, J = 8.0 Hz); 13C-
NMR (125 MHz, CDCl3) ꢀ ppm: 10.3, 21.9, 32.7, 67.3, 119.9,
120.2, 138.4, 151.0*, 158.0*, 169.0*; HRMS (ESI–TOF) m/z:
246.0357 [M+H]+ (calcd for C10H13ClNO2S, 246.0350).
centration of 500 ppm in the well. At least six wells were set up for
each test sample and control. After 24 h incubation, counts of static
and mobile J2 larvae were recorded and converted into percentages.
3. Anthelmintic Assay
Details of the experimental procedures were provided in proto-
col numbers 03-05-09 and 06-05-09, which were evaluated and
approved by the Honorary Animal Experimental Commission –
UdelaR (www.chea.udelar.edu.uy) under current regulations. The
effects of compounds 7 - 33 on the parasitant stage (L4) of N. bra-
siliensis were evaluated using a previously described method [21].
Sixty-day old Wistar rats were infected by subcutaneous injection
with 5000 N. brasiliensis L3 larvae (both animals and nematode
strains were maintained in the Animal Experimental Laboratory,
Facultad de Química, Universidad de la República. After 72 h, each
animal was sacrificed by cervical dislocation, the small intestine
was isolated, opened longitudinally and incubated at 37 °C in a
Baerman apparatus. The L4 parasites were collected and grown in
culture plates (containing 24 x 2 mL wells) on culture medium con-
taining antibiotic, serum and erythrocyte to which had been added
10 ꢃL of test sample or albendazole (as positive control) dissolved
in DMSO and diluted with an appropriate amount of this reagent in
order to give the required final concentration within the well. Nega-
tive control wells contained 10 ꢃL of DMSO instead of the test
sample. After 5 days in culture, live and dead larvae in each well
were counted with the aid of an inverted microscope (Nikon
TS100). A range of concentrations of selected test compounds was
assayed in order to obtain the corresponding concentration/activity
regression and the EC50 value.
10. General Method of Synthesis of Compounds 32 and 33
Compound 11 (75 mg, 0.32 mmol) was added to a suspension
of LiAlH4 (80 mg, 2.1 mmol) in anhydrous THF (50 mL) main-
tained under a nitrogen atmosphere and cooled in an ice bath. The
reaction mixture was stirred for 12 h, following which a saturated
solution of NH4Cl was poured onto the mixture and the whole ex-
tracted with EtOAc. The organic layer was dried over MgSO4, the
solvent was evaporated under reduced pressure, and the resulting
crude product subjected to preparative TLC and developed twice
with n-hexane:EtOAc (7:3) to afford 32 [13]. Compound 12 was
treated in a similar fashion to afford product 33. Compound 32 ([(6-
chloropyrazin-2-yl)sulfanyl] ethanol) was obtained as a yellow oil
1
in 29% yield: TLC Rf 0.22 (n-hexane–EtOAc, 1:1); H-NMR (500
MHz, CDCl3) ꢀ ppm: 3.33 (t, 2H, J = 6.0 Hz), 3.85 (t, 2H, J = 6.0
Hz), 8.16 (s, 1H), 8.31 (s, 1H); 13C-NMR (125 MHz, CDCl3) ꢀ
ppm: 33.3, 61.8, 139.1, 141.7, 149.0*, 156.7; HRMS (ESI–TOF)
m/z: 191.0031 [M+H]+ (calcd for C6H8ClN2OS, 191.0040). Com-
pound 33 (2,2'-[(pyrazin-2,6-diyl)bissulfandiyl] diethanol) was
obtained as an orange solid in 6% yield; TLC Rf 0.37 (n-hexane–
1
EtOAc, 1:1); H-NMR (500 MHz, CDCl3) ꢀ ppm: 3.27 (t, 4H, J =
7.0 Hz), 3.77 (t, 4H, J = 7.0 Hz), 8.06 (s, 2H). 13C-NMR (125 MHz,
CDCl3) ꢀ ppm: 31.5, 61.8, 138.5, 155.2; HRMS (ESI–TOF) m/z:
233.0415 [M+H]+ (calcd for C8H13N2O2S2, 233.0413).
4. Acute Toxicity Assay
Details of the experimental procedures were provided in proto-
col number 09-05-09, which was evaluated and approved by the
Biological Evaluations
Honorary Animal Experimental Commission
–
UdelaR
(www.chea.udelar.edu.uy) under current regulations. Toxicity was
evaluated according to OECD 425 guidelines [16] and involved oral
administration of 2000 mg/kg of the test compound to each of five
female Wistar rats. Variation in behavior was observed (Irwin test)
at 30 min, 1, 2, 4, 6, 24 and 48 h, and 7 and 14 days post-dose. The
body weights of the experimental animals were recorded the day
before oral administration and 7 and 14 days post-dose. After 14
days, the animals were sacrificed, submitted to necropsy and all
abnormalities registered. Evaluation of the LD50 value of the test
compound was performed using the OECD program AOT425
StatPgm, and was based on the long-term (14 day) data.
1. Anticholinesterasic Assay
A standard bioautographic method [15] was employed in which
TLC layers were loaded with the synthetic test compounds in de-
creasing concentration range, developed with the appropriate sol-
vent system and subsequently dried. The layers were then sprayed
with a solution containing 6.66 U/mL of electric eel AChE type V
(Sigma-Aldrich; product no. C 2888), re-dried and subsequently
incubated at 37 °C for 20 min in a moist atmosphere. Enzyme activ-
ity was detected by spraying the layers with a freshly prepared solu-
tion containing 1-naphthyl acetate (0.25%) in EtOH (1 vol.) and
Fast Blue B salt (0.25%) in water (4 vol.). Potential acetylcho-
linesterase inhibitors appeared as clear zones on a purple colored
background.
ACKNOWLEDGEMENTS
This study was supported by FAPESP as part of the Biota-
FAPESP Biodiversity Virtual Institute Program (www.biota.org.br)
through grant no. 03/02176-7 awarded to V.S.B. The authors also
wish to acknowledge FAPESP, CAPES and CNPq for fellowships,
and to thank Ximena Ures and María José Andina Alonzo (Univer-
sidad de la República-Uruguay) for the anthelmintic evaluation.
2. Nematostatic Assay
Compounds were assayed for their in vitro ability to immobilize
second-stage juveniles (J2) of the root-knot nematode M. incognita.
Juveniles were collected immediately after hatching from the eggs
[18, 19] and were employed in the assays less than 2 days after
emergence. A 20 ꢃL aliquot of an aqueous suspension containing
approximately 25 J2 was added to each 300 ꢃL well of a 96-well
polypropylene plate [20], together with the test sample or the posi-
tive control dissolved in aqueous Tween 80 or water (100 ꢃL), and
30 ꢃL of an aqueous solution containing a mixture of antibacterial
agents (3.0 mg/mL; Pentabiotico®, Fort Dodge, Brazil). Negative
control wells contained 100 ꢃL of 1% (w/v) aqueous Tween 80
solution instead of the test sample. For each of the test compounds,
appropriate dilutions were prepared in 1% (w/v) aqueous Tween 80
solution in order to obtain a final concentration of 500 ppm in each
well. For the positive control, a solution of aldicarb was prepared
by suspending 8.6 g of Temik 150 (containing 150 g of aldicarb/kg;
Rhône-Poulenc Agro-Brasil) in water and passing the stirred sus-
pension through filter paper: appropriate dilutions were prepared in
1% (w/v) aqueous Tween 80 solution in order to obtain a final con-
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publishers Web site
along with the published article.
REFERENCES
[1]
Villain, L.; Aribi, J.; Reversat, G.; Anthony, F. A high-throughput method
for early screening of coffee (Coffea spp.) genotypes for resistance to root-
knot nematodes (Meloidogyne spp.). Eur. J. Plant Pathol., 2010, 128(4),
451-458.
[2]
[3]
Molento, M.B. Parasite control in the age of drug resistance and changing
agricultural practices. Vet. Parasitol., 2009, 163(3), 229-234.
Nari, A.; Salles, J.; Gil, A.; Waller, P.J.; Hansen, J.W. The prevalence of
anthelmintic resistance in nematode parasites of sheep in southern Latin
America: Uruguay. Vet. Parasitol., 1996, 62(3-4), 213-222.

3430 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
Valli et al.
[4]
Waller, P.J.; Echevarria, F.; Eddi, C.; Maciel, S.; Nari, A.; Hansen, J.W. The
prevalence of anthelmintic resistance in nematode parasites of sheep in
southern Latin America: general overview. Vet. Parasitol., 1996, 62(3-4),
181-187.
Jabbar, A.; Iqbal, Z.; Kerboeuf, D.; Muhammad, G.; Khan, M.N.; Afaq, M.
Anthelmintic resistance: the state of play revisited. Life Sci., 2006, 79(26),
2413-2431.
Taylor, M.A.; Hunt, K.R.; Goodyear, K.L. The effects of stage-specific
selection on the development of benzimidazole resistance in Haemonchus
contortus in sheep. Vet. Parasitol., 2002, 109(1-2), 29-43.
Gill, J.H.; Kerr, C.A.; Shoop, W.L.; Lacey, E. Evidence of multiple mecha-
nisms of avermectin resistance in Haemonchus contortus–comparison of se-
lection protocols. Int. J. Parasitol., 1998, 28(5), 783-789.
Viegas Jr., C.; Bolzani, V.S.; Furlan, M.; Barreiro, E.J.; Young, M.C.M.;
Tomazela, D.; Eberlin, M.N. Further bioactive piperidine alkaloids from the
flowers and green fruits of Cassia spectabilis. J. Nat. Prod., 2004, 67(5),
908-910.
Viegas Jr., C.; Bolzani, V.S.; Pimentel, L.S.B.; Castro, N.G.; Cabral, R.F.;
Costa, R.S.; Floyd, C.; Rocha, M.S.; Young, M.C.M.; Barreiro, E.J.; Fraga,
C.A.M. New selective acetylcholinesterase inhibitors designed from natural
piperidine alkaloids. Bioorg. Med. Chem., 2005, 13(13), 4184-4190.
Viegas Jr., C.; Danuello, A.; Bolzani, V.S.; Barreiro, E.J.; Fraga, C.A.M.
Molecular hybridization: a useful tool in the design of new drug prototypes.
Curr. Med. Chem., 2007, 14(17), 1829-1852.
Carvalho, A.S.; Fraga, C.A.M.; Barreiro, E.J. Synthesis of condensed tri-
cyclic pyrazolo[3,4-b]thieno[2,3-d]pyridine and related isostere derivatives.
J. Heterocycl. Chem., 1996, 33(2), 309-313.
methanesulfonamido-1-indanone derivatives. J. Med. Chem., 1995, 38(25),
4897-4905.
Branowska, D.; Buczek, I.; Kaliꢄska, K.; Nowaczyk, J.; Rykowski, A. S-
Transalkylation/ring closing metathesis as a route to azathiamacrocycles in-
corporating 2,2'-bipyridine subunits. Tetrahedron Lett., 2005, 46(49), 8539-
8541.
Avetisyan, A.A.; Aleksanyan, I.L. Synthesis of derivatives of (4-methyl-2-
quinolylthio)acetic and (4-methyl-2-quinolylthio)propionic acids. Chem.
Heterocycl. Compd., 2005, 41(3), 350-353.
[13]
[5]
[6]
[7]
[8]
[14]
[15]
[16]
Marston, A.; Kissling, J.; Hostettmann, K. A rapid TLC bioautographic
method for the detection of acetylcholinesterase and butyrylcholinesterase
inhibitors in plants. Phytochem. Anal., 2002, 13(1), 51-54.
Organization for Economic Cooperation and Development (OECD). Guide-
lines for testing chemicals. Acute oral toxicity-up and down procedure. 2001,
425, http://www.oecd.org/dataoecd/17/51/1948378.pdf, (Acessed May 30,
2011).
[17]
[18]
Perrin, D.D.; Armarego, W.L.F. Purification of laboratory chemicals, 3rd ed.;
Pergamon Press: Oxford, 1988.
[9]
Hussey, R.S.; Barker, K.R. Comparison of methods of collecting inocula of
Meloidogyne spp, including a new technique. Plant Dis. Rep., 1973, 57(12),
1025-1028.
[10]
[11]
[12]
[19]
[20]
[21]
Boneti, J.I.S.; Ferraz, S. Modificação do método de Hussey e Barker para
extração de ovos de Meloidogyne exigua de cafeeiro. Fitopatol. Bras., 1981,
6(3), 553.
Amaral, D.R.; Oliveira, F.E.D.; Oliveira, D.F.; Campos, V.P. Purification of
two substances from bulbs of onion (Allium cepa L.) with nematicidal activ-
ity against Meloidogyne exigua Goeldi. Nematology, 2003, 5(6), 859-864.
Gordon, S.; Costa, L.; Incerti, M.; Manta, E.; Saldaña, J.; Dominguez, L.;
Mariezcurrena, R.; Suescun, L. Synthesis and in vitro anthelmintic activity
against Nippostrongylus brasiliensis of new 2-amino-4-hydroxy-delta-
valerolactam derivatives. Farmaco, 1997, 52(10), 603-608.
Li, C.S.; Black, W.C.; Chan, C.C.; Fordhutchinson, A.W.; Gauthier, J.Y.;
Gordon, R.; Guay, D.; Kargman, S.; Lau, C.K.; Mancini, J.; Ouimet, N.;
Roy, P.; Vickers, P.; Wong, E.; Young, R.N.; Zamboni, R.; Prasit, P. Cy-
clooxygenase-2 inhibitors. Synthesis and pharmacological activities of 5-
Received: March 14, 2011
Revised: May 31, 2011
Accepted: June 02, 2011