Piperazine N-substituted naphthyridines, pyridothienopyrimidines and pyridothienotriazines: New antiprotozoals active against Philasterides dicentrarchi

Article abstract of DOI:10.1016/S0223-5234(03)00032-1

New antiprotozoals active against Philasterides dicentrarchi, the causative agent of scuticociliatosis in farmed turbot and Black Sea bass-bream, have been synthesised and tested. The most active compounds posses a piperazine ring, generally N-bonded to t

Full text of DOI:10.1016/S0223-5234(03)00032-1

European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
www.elsevier.com/locate/ejmech
Original article
Piperazine N-substituted naphthyridines, pyridothienopyrimidines
and pyridothienotriazines: new antiprotozoals active against
Philasterides dicentrarchi
Jose´ M. Quintela a, Carlos Peinador a, Liliana Gonza´lez a, Rau´l Iglesias b,
b
b
b
Anabel Parama´ , Francisca Alvarez , Manuel L. Sanmart´ın , Ricardo Riguera *
c,
´
a Departamento de Qu´ımica Fundamental e Industrial, Facultad de Ciencias, Universidad de La Coruna, La Coruna 15071, Spain
˜
˜
b Laboratorio de Parasitologia, Instituto de Investigacio´n y Ana´lisis alimentarios, Universidad de Santiago, Santiago de Compostela 15706, Spain
c Departamento de Qu´ımica Orga´nica, Facultad de Quimica, Instituto de Acuicultura, Universidad de Santiago, Santiago de Compostela 15706, Spain
Received 16 October 2002; received in revised form 13 January 2003; accepted 24 January 2003
Abstract
New antiprotozoals active against Philasterides dicentrarchi, the causative agent of scuticociliatosis in farmed turbot and Black
Sea bass-bream, have been synthesised and tested. The most active compounds posses a piperazine ring, generally N-bonded to the
heterocycle, and are the 1,8-naphthyridines, 2f and 5o, the pyridothienopyrimidine (7), and the pyridothienotriazines, 8, 9, 12d, 12f,
12h, 12m and 12k. Pyridothienotriazine (12k) presents the same activity (Lethal Dose, LDꢀ
0.8/1.5 mg Lꢁ1) as the well-known
antiparasitics niclosamide and oxyclozanide.
/
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 1,8-Naphthyridine; Pyridothienopyrimidine; Pyridothienotriazine; Antiprotozoal
1. Introduction
include Uronema marinum, Uronema nigricans, Ano-
phryoides haemophila and Mesanophrys spp. [1ꢂ4]. In
/
The study of the diseases and disorders found in
cultivated fish, crustaceans and molluscs is particularly
important due to the ease with which pathogenic agents
are transmitted in closed water tanks and the great
economic losses incurred from the morbidity and
mortality of cultivated marine animals.
In general, the incidence of different parasitic diseases
in farm animals is so high that the antiparasitic drug
sector is the most important in the animal health market
and a very large proportion of the commercial demand
for these compounds is related to livestock.
In the field of aquaculture the name scuticociliatosis is
well known and is used to identify those diseases of fish,
crustaceans and molluscs that are caused by histopha-
gous ciliates of the order Scuticociliatida. The most
important pathogenic species belonging to this order
the last few years there have been reports of several fatal
outbreaks of systemic scuticociliatosis due to Philaster-
ides dicentrarchi in farmed sea bass-bream and turbot
[5,6]. In the latter case, the incidence of this disease
appears to have increased to the extent that it constitutes
one of the major parasitic infections in turbot farms and
is the cause of significant commercial losses.
For these reasons, screening programs and other
efforts to find drugs that are effective against this
parasite are especially important. Recently, the in vitro
screening of a large number of known pharmaceuticals,
selected on the basis of their therapeutic activity in
related processes, has been reported [7]. However, really
effective control measures remain unknown.
In the course of a study aimed at the discovery of new
compounds to combat scuticociliatosis, we centred our
efforts on the identification of structural characteristics
that could be associated with the desired activity and
that could eventually lead to the discovery, design and
preparation of effective antiprotozoals.
* Corresponding author.
E-mail address: ricardo@usc.es (R. Riguera).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00032-1

266
J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
In this paper we will show that 1,8-naphthyridines,
Pyridothienopyrimidine (7) [12] and pyridothienotria-
zines, 8 and 9 [13] are previously known compounds and
were obtained following methods described in the
literature. In the preparation of pyridothienotriazines,
pyridothienotriazines and pyridotrienodiazines N-
bonded to a piperazine residue, are antiprotozoal agents
active in vitro against P. dicentrarchi.
12aꢂp, which are disubstituted at positions 4 and 7, we
/
decided to introduce the first substituent in the starting
material 10 and use the corresponding 4-chloro-deriva-
tive 11 as the key intermediate for the introduction of
the second substituent by halide displacement with
nucleophiles.
Intermediate 11 was prepared by diazotisation of 3-
amino-2,5-dicyano-6-substituted thieno[2,3-b]pyridines
(10) [14] and spontaneous intramolecular condensation
of the diazonium ion with the adjacent nucleophilic
function (Fig. 3).
All the compounds gave satisfactory elemental ana-
lyses and spectral data (IR, MS, and 1H- and 13C-NMR)
that are consistent with the structures proposed. Details
of the synthesis and corresponding experimental data
for these compounds are included in the experimental
part.
2. Chemistry
N-heteroaromatic carbaldehydes are extensively used
as versatile synthetic building blocks for the preparation
of condensed heterocycles [8]. On the basis of this
information, we devised synthetic procedures to convert
ortho-aminoaldehyde (1) into 1,8-naphthyridines, 2 and
5, which are appropriately substituted at position 7.
Condensation of the heterocyclic ortho-aminoaldehyde
(1) [9] with aromatic methyl ketones under catalytic
alkaline conditions (ethanolic potassium hydroxide)
yielded the expected Friedlander products 2aꢂn as
shown in Fig. 1.
On the other hand, condensation of aminoaldehyde
/
¨
(1) with ethyl cyanoacetate [10] produced 2-ethoxy-3,6-
(3),
dicyano-7-hydroxy-4-phenyl-1,8-naphthyridine
which is easily converted to the chloroderivative 4 by
reaction with phosphorus pentachloride.
The 7-chloro-3,6-dicyano-1,8-naphthyridine (4) was
used as a starting material for the preparation of a series
3. Pharmacology
of 7-substituted 1,8-naphthyridines (5aꢂo) by nucleo-
/
In order to ensure the reliability of the test results for
the antiprotozoal activity of our compounds against P.
dicentrarchi, it was of fundamental importance that the
cultivated ciliates remained infective throughout the
experiments. To this end, ciliates were harvested by
collecting ascitic fluid from the body cavity of naturally
infected turbot. The ciliates were then maintained under
the culture conditions described by Bernard and Fenchel
[15] but with autoclaved Vibrio anguillarum as food.
Under these conditions, P. dicentrarchi retains its
capacity to induce scuticociliatosis in experimentally
infected turbot.
philic halide displacement with nucleophiles (Fig. 2).
Pyrido[2,3-d]pyrimidine (6) is a new compound and
was obtained according to published procedures [11].
In addition, the tests were performed in two different
PBS conditions and also in
media: the standard DMSOꢂ
/
DMSOꢂfiltered sea water. The results obtained in sea
/
water are particularly important because they automa-
tically exclude the possibility of inactivation of the test
substance under marine conditions. In fact, a compound
found to be effective in vitro by this procedure can also
be expected to be effective in bath administration to
infected fish or other organisms.
Drugs against Philasterides must be able to comple-
tely eliminate the ciliates because, due to their extremely
high reproduction rate in the fish host, the survival of as
few as one to two ciliates per host is enough to cause a
second bout of infection. In this particular case, LD99 is
a better index of effectivity than LD50 and accordingly,
we have expressed our results as LD99: the minimum
lethal concentration required to kill 99% of the ciliates.
Fig. 1. Synthetic scheme and structures of the 3-cyano-2-ethoxy-4-
phenyl-7-substituted-1,8-naphthyridines 2aꢂn.
/

J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ
/
275
267
Fig. 2. Synthetic scheme and structures of the 3,6-dicyano-2-ethoxy-4-phenyl-7-substituted-1,8-naphthyridines 5aꢂo.
/
4. Results and discussion
When the 1,8-naphthyridines, 2aꢂ
to in vitro tests for antiparasitic activity towards the
ciliate P. dicentrarchi in PBS and in sea water (Table 1),
we found that one of the compounds (2f) was active in
both solvent systems, showing Lethal Dose (LD) values
of 50 and 12.5 mg Lꢁ1, respectively. Similar testing on
naphthyridine system of a piperazine substituent and
that this unit should preferably have a free NH group.
We attempted to estimate the relative contributions of
the different parts of 1,8-naphthyridine and piperazine
molecules in the global activity of these compounds. To
this end, we tested some simple N-phenylpiperazines
and found that only those carrying electron-withdraw-
ing substituents (i.e. 2-Cl; 3-Cl; 4-Cl and 3-CF3) show
any activity and, in these cases, it is only moderate
/
n, were submitted
1,8-naphthyridines, 5aꢂo, revealed that only compound
/
5o was active at a similar dose, clearly indicating that the
activity is associated with the presence on the 1,8-
(LDꢀ
/
100 mg Lꢁ1). Even this low level of activity is not
observed in the unsubstituted N-phenylpiperazine either
Fig. 3. Synthetic scheme and structures of the 8-cyano-4,7-disubstituted-pyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazines 12aꢂp.
/

268
J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
Table 1
In vitro activity of selected compounds against the protozoal P.
dicenterarchi
ing that free access to the NH group plays a crucial role
in the activity of these compounds.
Replacing the naphthyridine ring of 2f and 5o, with
other electron-poor nitrogen heterocycles such as the
pyridopyrimidine (6) (Fig. 4) produced moderately
Entry
Compound
Solvent A
Solvent B
1
2
3
4
5
6
7
8
9
10
2f
50.0
25.0
12.5
6.1
12.5
12.5
12.5
3.1
active compound (LDꢀ
100 mg Lꢁ1), only but better
/
5o
7
results were obtained in the pyridothienopyrimidine and
the pyridothienotriazine systems. In fact a good level of
activity was found in those compounds bearing the
piperazine ring, i.e. pyridothienodiazine (7) and pyri-
dothienotriazines, 8 and 9.
Comparison of compounds 8 and 9 indicate that
substitution of the ethoxy group at position 7 by a
phenyl and of the cyano group at position 8 by
hydrogen, produced a 4-fold increase of the LD values.
Replacement in the pyridothienotriazine (8) of the
phenyl group at position 9 by hydrogen and of the
ethoxy group at position 7 by nitrogen nucleophiles
8
9
25.0
50.0
25.0
12.5
25.0
0.8
25.0
25.0
12.5
50.0
25.0
1.5
12d
12f
12m
12h
12k
The LD (in mg Lꢁ1) are measured in DMSOꢂ
in sea water (solvent B).
/PBS (solvent A) and
when the phenyl ring is separated from the piperazine by
one, two or three carbons or when the piperazine system
is linked to the nitrogen through an acyl group.
produced a new series of compounds 12aꢂp, which all
/
had significant antiprotozoal activity particularly 12k,
the most active compound found in this work. The
structures of representative piperazine-substituted anti-
protozoals described in this work are presented in Fig. 4
and the LD values for a selection of the most active
compounds (i.e. with LD values of 25 mg Lꢁ1 or less)
are given in Table 1.
All this suggests that the presence of the electron-poor
heteroaromatic ring in compounds 2 and 5 is essential
for strong antiprotozoal activity. This is consistent with
the activity of the well-known antibiotic norfloxazine
(LDꢀ
/
50 mg Lꢁ1 in both solvents) (Fig. 4) while its
analogue lomefloxazine is completely inactive, suggest-
Fig. 4. Structures of representative piperazine-substituted antiprotozoals described in this work.

J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ
/
275
269
5. Conclusions
t, Jꢀ
7.42 (1H, d, Jꢀ
8.05 (1H, d, Jꢀ
C6H4). 13C-NMR (CDCl3): dꢀ
(OCH2); 99.5 (CN); 114.2 (C-3); 116.8; 120.6; 124.6;
129.0; 129.2; 130.0; 130.2; 131.6; 132.8; 133.0; 135.1;
137.0; 148.4; 155.4; 158.4; 161.7; 162.6. MS (EI, m/z, %):
396 [Mꢃ, 49]. Anal. C23H16N4O3 (C, H, N).
/
7.1 Hz, CH3); 4.78 (2H, q, Jꢀ
/
7.1 Hz, OCH2);
/
8.5 Hz, H-6); 7.74 (8H, m, C6H5, C6H4);
8.5 Hz, H-5); 8.08 (1H, d, Jꢀ7.8 Hz,
14.3 (CH3); 64.3
In conclusion, our results demonstrate that 1,8-
naphthyridines, pyridothienopyrimidines and pyri-
dothienotriazines incorporating a piperazine group
(that keeps a free NH), is directly related to the
antiprotozoal activity of the compounds against the
ciliate P. dicentrarchi. Specific examples of effective new
antiparasitic compounds with these structural charac-
teristics include pyridothienopyrimidine (7), pyridothie-
notriazines 8, 9, 12d, 12f, 12h, 12m and, in particular,
/
/
/
6.1.1.3. 3-Cyano-2-ethoxy-7-(2-hydroxyphenyl)-4-
phenyl-1,8-naphthyridine (2b). Reaction time: 3 h.
12k*
/
whose LD value (0.8 mg Lꢁ1 in PBS and 1.5 mg
Recrystallised from ethanol. M.p.: 259ꢂ
69%; IR (KBr, cmꢁ1): nꢀ
2220 (CN); 1590; 1565; 1420;
1330; 1235; 1160; 750. 1H-NMR (CDCl3): dꢀ
1.59 (3H,
t, Jꢀ7.2 Hz, CH3); 4.76 (2H, q, Jꢀ7.2 Hz, OCH2);
6.93ꢂ7.13 (2H, m, C6H5,); 7.39ꢂ7.93 (8H, m, C6H5,
C6H4, H-6); 8.06 (1H, d, Jꢀ8.8 Hz, H-5); 14.90 (1H, s,
OH). 13C-NMR (CDCl3): dꢀ
14.3 (CH3); 64.3 (OCH2);
/
261 8C. Yield:
Lꢁ1 in sea water) is equal to those of other known
antiparasitics such as niclosamide and oxyclozanide (0.8
mg Lꢁ1) [7]. Studies aimed at improving this activity
and the development of other piperazine-substituted
heterocycles are currently in progress.
/
/
/
/
/
/
/
/
114.3 (CN); 115.8; 116.6; 119.0; 119.1; 127.3; 129.1;
129.3; 130.3; 132.8; 133.4; 135.2; 137.3; 153.3; 157.7;
161.7; 162.6; 166.1. MS (FAB, m/z, %): 368 [(MHꢃ),
58]. Anal. C23H17N3O2 (C, H, N).
6. Experimental
6.1. Chemistry
All melting points were measured on a Buchi 510
¨
instrument and are uncorrected. IR spectra (potassium
6.1.1.4. 7-(3-Aminophenyl)-3-cyano-2-ethoxy-4-phenyl-
1,8-naphthyridine (2c). Reaction time: 20 h. Recrystal-
bromide) were recorded on a Perkinꢂ
/
Elmer 383 spectro-
lised from ethanol. M.p.: 276ꢂ
(KBr, cmꢁ1): nꢀ
3350 (NH2); 3000; 2240 (CN); 1590;
1340; 1170; 780. 1H-NMR (CDCl3): dꢀ
1.56 (3H, t, Jꢀ
7.1 Hz, CH3); 3.60 (2H, br s, NH2); 4.82 (3H, q, Jꢀ7.1
Hz, OCH2); 6.81ꢂ7.68 (9H, m, C6H5, Py); 7.77 (1H, d,
Jꢀ8.6 Hz, H-6); 7.99 (1H, d, Jꢀ
8.6 Hz, H-5). 13C-
NMR (CDCl3): dꢀ14.4 (CH3); 64.1 (OCH2); 98.4 (C-
/
277 8C. Yield: 67%; IR
photometer. 1H- and 13C-NMR spectra (200 and 50
MHz) were recorded on a Bruker AC200F spectrometer.
Mass spectra were obtained on a VG4 spectrometer.
Microanalyses for C, H and N were performed by the
Elemental Analysis General Service of the University of
/
/
/
/
/
/
/
La Coruna. Analyses indicated by symbols of the
˜
/
elements or functions were within 9
theoretical values. Silica gel HF254ꢃ366 for thin layer
chromatography and silica gel 60 (230ꢂ400 mesh) for
flash chromatography were used as purchased from
Merck. All reagents were commercial-grade chemicals
from freshly opened containers.
/
0.4% of the
3); 114.6 (CN); 116.6; 118.2; 129.0; 129.3; 129.7; 130.1;
133.2; 136.9; 139.0; 147.0; 156.1; 158.2; 162.7; 162.8. MS
(EI, m/z, %): 366 [Mꢃ, 100]. Anal. C23H18N4O (C, H,
N).
/
6.1.1.5. 3-Cyano-2-ethoxy-7-(4-fluorophenyl)-4-phenyl-
1,8-naphthyridine (2d). Reaction time: 10 h. Recrystal-
6.1.1. 3-Cyano-2-ethoxy-4-phenyl-1,8-napthyridines
lised from ethanol. M.p.: 205ꢂ
(KBr, cmꢁ1): nꢀ
2230 (CN); 1580; 1500; 1380; 1210;
810. 1H-NMR (CDCl3): dꢀ
1.56 (3H, t, Jꢀ7.1 Hz,
CH3); 4.81 (2H, q, Jꢀ7.1 Hz, OCH2); 7.13ꢂ7.27 (2H,
m, C6H4F); 7.46ꢂ7.65 (5H, m, C6H5); 7.75 (1H, d, Jꢀ
8.5 Hz, H-6); 8.02 (1H, d, Jꢀ8.5 Hz, H-5); 8.17ꢂ8.27
(2H, m, C6H4F). 13C-NMR (CDCl3): dꢀ
14.3 (CH3);
/
206 8C. Yield: 79%; IR
(2aꢂ
/
m)
/
/
/
6.1.1.1. General procedure. A solution of 1 (0.75 mmol),
and the methyl ketone (or acetonitrile in the case of 2n)
(0.90 mmol) and a catalytic amount of 10% ethanolic
potassium hydroxide in ethanol (10 mL) was refluxed
until all starting material had disappeared as checked by
TLC. After cooling, the precipitate was collected by
filtration and recrystallised from a suitable solvent or
purified by flash chromatography.
/
/
/
/
/
/
/
64.2 (OCH2); 98.6 (CN); 114.4 (C-3); 115.7; 116.1; 116.5;
118.1; 129.0; 129.3; 130.1; 130.2; 133.1; 134.2; 137.2;
155.8; 158.2; 161.5; 162.1; 164.6 (d, JCÃF
ꢀ253 Hz). MS
/
(EI, m/z, %): 369 [Mꢃ, 55]. Anal. C23H16N3OF (C, H,
N).
6.1.1.2. 3-Cyano-2-ethoxy-7-(2-nitrophenyl)-4-phenyl-
1,8-naphthyridine (2a). Reaction time: 24 h. Recrystal-
6.1.1.6. 3-Cyano-2-ethoxy-7-(3,4-dimethoxyphenyl)-4-
phenyl-1,8-naphthyridine (2e). Reaction time: 1 h.
lised from ethanol. Melting point (m.p.) 271ꢂ
Yield: 68%; IR (KBr, cmꢁ1): nꢀ
2240 (CN); 1585; 1570;
1630; 1330; 1315; 760. 1H-NMR (CDCl3): dꢀ
1.53 (3H,
/
272 8C.
/
Recrystallised from ethanol. M.p.: 228ꢂ
/
229 8C. Yield:
/
68%; IR (KBr, cmꢁ1): nꢀ
/3000; 2240 (CN); 1590; 1510;

270
J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
1
1275; 1160; 1040. H-NMR (CDCl3): dꢀ
Jꢀ7.1 Hz, CH3); 3.96 (3H, s, OCH3); 4.06 (3H, s,
OCH3); 4.82 (2H, q, Jꢀ7.1 Hz, OCH2); 7.98 (1H, d,
Jꢀ8.4 Hz, H-2); 7.46ꢂ
7.98 (9H, m, C6H5). 13C-NMR
(CDCl3): dꢀ14.4 (CH3); 56.0 (OCH3); 56.1 (OCH3);
/1.56 (3H, t,
(99.5/0.5). Yield: 76%; m.p.: 221ꢂ
cmꢁ1): nꢀ
2220 (CN); 1585; 1570; 1445; 1380; 1310;
1215; 1025; 800. 1H-NMR (CDCl3): dꢀ
1.58 (3H, t, Jꢀ
7.1 Hz, CH3); 4.83 (2H, q, Jꢀ7.1 Hz, OCH2); 7.45ꢂ7.54
(3H, m, Py, C6H5); 7.60ꢂ7.66 (3H, m, C6H5); 7.83 (1H,
d, Jꢀ8.8 Hz, H-6); 8.10 (1H, d, Jꢀ8.8 Hz, H-5); 8.56ꢂ
8.62 (1H, m, Py); 8.73ꢂ8.77 (1H, m, Py); 9.35ꢂ9.36 (1H,
m, Py). 13C-NMR (CDCl3): dꢀ
4.3 (CH3); 64.3 (OCH2);
117.1; 118.2; 123.7; 129.1; 129.3; 130.3; 133.0; 133.8;
135.3; 135.7; 137.7; 149.1; 151.3; 158.4; 160.2. EM
(FAB, m/z, %): 353 [(MHꢃ), 100]. Anal. C22H16N4O
(C, H, N).
/
222 8C. IR (KBr,
/
/
/
/
/
/
/
/
/
/
/
64.1 (OCH2); 98.1 (C-3); 110.9; 114.6 (CN); 116.2; 118.1;
121.2; 129.0; 129.3; 130.1; 130.8; 133.2; 136.8; 149.4;
151.5; 155.9; 158.1; 162.2; 162.8. MS (EI, m/z, %): 411
[Mꢃ, 100]. Anal. C25H21N3O3 (C, H, N).
/
/
/
/
/
/
6.1.1.7. 3-Cyano-2-ethoxy-4-phenyl-7-(4-
piperazinophenyl)-1,8-naphthyridine (2f). Reaction time:
48 h. Recrystallised from ethanol. M.p.: 216ꢂ
Yield: 50%; IR (KBr, cmꢁ1): nꢀ
3240 (NH); 2220 (CN);
1580; 1320; 1235; 805. 1H-NMR (CDCl3): dꢀ
1.55 (3H,
t, Jꢀ7.1 Hz, CH3); 1.76 (1H, br s NH); 3.06ꢂ3.10 (4H,
m, NCH2); 3.31ꢂ
7.1 Hz, OCH2); 7.01, 7.19 (4H, AA?XX? system, Jꢀ
Hz, C6H4); 7.46ꢂ
8.7 Hz, H-6); 7.92 (1H, d, Jꢀ
(CDCl3): dꢀ14.3 (CH3); 45.8 (NCH2); 48.9 (NCH2);
63.8 (OCH2); 97.2 (CN); 114.7 (C-3); 116.0; 117.6; 127.9;
128.9; 129.3; 130.0; 133.4; 136.5; 153.3; 156.2; 157.9;
162.2; 162.8. MS (EI, m/z, %): 435 [Mꢃ, 45]. Anal.
C27H25N5O (C, H, N).
/
218 8C.
/
6.1.1.11. 3-Cyano-2-ethoxy-4-phenyl-7-(4-pyridyl)-1,8-
naphthyridine (2j). Reaction time: 2.5 h. Purified by
/
/
/
flash chromatography eluting with dihloromethaneꢂ
ethanol (99.5/0.5). Yield: 84%; m.p.: 227ꢂ228 8C. IR
(KBr, cmꢁ1): nꢀ
2220 (CN); 1590; 1565; 1385; 1325;
1165; 1020; 800. 1H-NMR (CDCl3): dꢀ
1.58 (3H, t, Jꢀ
7.1 Hz, CH3); 4.83 (2H, q, Jꢀ7.1 Hz, OCH2); 7.47ꢂ7.52
(2H, m, C6H5); 7.63ꢂ7.66 (3H, m, C6H5); 7.84 (1H, d,
Jꢀ8.8 Hz, H-6); 8.06ꢂ8.14 (3H, m, Py, H-5); 8.80ꢂ8.83
(2H, m, Py). 13C-NMR d (CDCl3): dꢀ
14.3 (CH3); 64.4
/
/
3.35 (4H, m, NCH2); 4.81 (2H, q, Jꢀ
/
/
/9.1
/
/
7.61 (5H, m, C6H5); 7.74 (1H, d, Jꢀ
/
/
/
/
8.7 Hz, H-5). 13C-NMR
/
/
/
/
/
/
/
/
(OCH2); 114.1 (CN); 117.7; 118.3; 121.9; 129.1; 129.3;
130.4; 132.9; 135.3; 139.9; 145.2; 150.6; 158.4; 158.4;
160.0. MS (FAB, m/z, %): 353 [(MHꢃ), 100]. Anal.
C22H16N4O (C, H, N).
6.1.1.8. 3-Cyano-2-ethoxy-4-phenyl-7-(2-thienyl)-1,8-
naphthyridine (2g). Reaction time: 20 min. Recrystal-
lised from ethanol. M.p.: 225ꢂ
(KBr, cmꢁ1): nꢀ
3050; 2220 (CN); 1595; 1415; 1315;
1205; 800; 700. H-NMR (CDCl3): dꢀ1.55 (3H, t, Jꢀ
7.1 Hz, CH3); 7.81 (2H, q, Jꢀ7.1 Hz, OCH2); 7.15ꢂ7.20
(1H, m, thienyl); 7.45ꢂ7.69 (7H, m, C6H5, thienyl, H-6);
7.83ꢂ
7.93 (2H, m, thienyl, H-5). 13C-NMR (CDCl3):
dꢀ14.4 (CH3); 64.1 (OCH2); 98.1 (CN); 114.5 (C-3);
116.5; 117.2; 128.1; 128.4; 129.0; 129.3; 130.1; 130.7;
133.1; 136.9; 143.6; 155.9; 157.3; 157.9; 168.9. MS (EI,
m/z, %): 357 [Mꢃ, 100]. Anal. C21H15N3OS (C, H, N).
/
226 8C. Yield: 73%; IR
6.1.1.12. 3-Cyano-2-ethoxy-7-(3-indolyl)-4-phenyl-1,8-
naphthyridine (2k). Reaction time: 24 h. Recrystallised
/
1
/
/
from ethanol. M.p.: 261ꢂ
/
262 8C. Yield: 45%; IR (KBr,
/
/
cmꢁ1): nꢀ
/
3250 (NH); 2220 (CN); 1600; 1580; 1530;
/
1
1210; 800. H-NMR (CDCl3): dꢀ
Hz, CH3); 4.69 (2H, q, Jꢀ7.1 Hz, OCH2); 7.22ꢂ
(2H, m, C6H4); 7.47ꢂ7.70 (8H, m, C6H4, C6H5, H-6);
7.97 (1H, d, Jꢀ8.8 Hz, H-5); 8.43 (1H, s, NH); 8.78ꢂ
8.82 (1H, m, H-2?). 13C-NMR (CDCl3): dꢀ
14.6 (CH3);
/
1.48 (3H, t, Jꢀ
/7.1
/
/
/7.26
/
/
/
/
/
63.2 (OCH2); 95.5 (CN); 112.2; 114.5 (C-3); 114.9; 115.1;
118.5; 121.2; 122.5; 122.6; 125.5; 128.5; 129.0; 129.5;
130.5; 133.2; 135.2; 137.2; 155.8; 157.3; 161.1; 162.1. MS
(EI, m/z, %): 390 [Mꢃ, 93]. Anal. C25H18N4O (C, H,
N).
6.1.1.9. 3-Cyano-2-ethoxy-4-phenyl-7-(2-pyridyl)-1,8-
naphthyridine (2h). Reaction time: 20 min. Recrystal-
lised from ethanol. M.p.: 226ꢂ
(KBr, cmꢁ1): nꢀ
2970; 2220 (CN); 1575; 1380; 1330;
1160; 1020. H-NMR (CDCl3): dꢀ1.53 (3H, t, Jꢀ7.1
Hz, CH3); 4.77 (2H, q, Jꢀ7.1 Hz, OCH2); 7.31ꢂ7.60
(6H, m, C6H5, Py); 7.78ꢂ7.87 (1H, m, Py); 7.99 (1H, d,
Jꢀ8.7 Hz, H-6); 8.44 (1H, d, Jꢀ8.7 Hz, H-5); 8.66ꢂ
8.71 (2H, m, Py). 13C-NMR (CDCl3): dꢀ
14.2 (CH3);
/
228 8C. Yield: 78%; IR
/
1
/
/
/
/
6.1.1.13. 3-Cyano-2-ethoxy-4-phenyl-7-(2-pirrolyl)-1,8-
naphthyridine (2l). Reaction time: 30 min. Recrystal-
/
/
/
/
lised from ethanol. M.p.: 180ꢂ
(KBr, cmꢁ1): nꢀ
3640 (NH); 2225 (CN); 1610; 1560;
1330; 1220; 810; 715. 1H-NMR (CDCl3): dꢀ
1.54 (3H, t,
Jꢀ7.1 Hz, CH3); 4.73 (2H, q, Jꢀ7.1 Hz, OCH2); 6.34ꢂ
6.38 (1H, m, C4H3NH); 6.94 (1H, broad singlet, NH);
7.05 (1H, s, C4H3NH); 7.43ꢂ7.61 (6H, m, C6H5, H-6);
7.72 (1H, d, Jꢀ8.7 Hz, H-5); 10.3 (1H, s, C4H3NH).
13C-NMR (CDCl3): dꢀ
14.3 (CH3); 63.9 (OCH2); 97.0
/
182 8C. Yield: 80%; IR
/
/
64.0 (OCH2); 98.9 (C-3); 114.3 (CN); 117.6; 118.5; 122.7;
124.9; 128.9; 129.2; 130.0; 132.9; 136.8; 137.0; 149.1;
154.5; 155.4; 158.2; 161.0; 162.4. MS (EI, m/z, %): 352
[Mꢃ, 49]. Anal. C22H16N4O (C, H, N).
/
/
/
/
/
/
6.1.1.10. 3-Cyano-2-ethoxy-4-phenyl-7-(3-pyridyl)-1,8-
naphthyridine (2i). Reaction time: 3 h. Purified by flash
/
(CN); 111.2; 112.0; 114.7 (C-3); 115.7; 117.0; 122.9;
128.9; 129.3; 130.0; 130.7; 133.2; 136.3; 154.8; 156.1;
chromatography eluting with dichloromethaneꢂethanol
/

J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ
/
275
271
157.8; 163.0. MS (EI, m/z, %): 340 [Mꢃ, 100]. Anal.
C21H16N4O (C, H, N).
THF (15 mL) until the starting material had disap-
peared (TLC). The solvent was removed under reduced
pressure and the residue recrystallised from ethanol.
6.1.1.14. 7-(2,4-Dimethylthiazol-5-yl)-3-cyano-2-
ethoxy-4-phenyl-1,8-naphthyridine (2m). Reaction time:
6.1.3.2. 7-(4-(4-Acetylphenyl)piperazino)-3,6-dicyano-
2-ethoxy-4-phenyl-1,8-naphthyridine (5a). Reaction
20 min. Recrystallised from ethanol. M.p.: 235ꢂ
Yield: 71%; IR (KBr, cmꢁ1): nꢀ
2230 (CN); 1600; 1380;
1330; 810. H-NMR (CDCl3): dꢀ1.55 (3H, t, Jꢀ7.1
Hz, CH3); 2.73 (3H, s, CH3); 2.75 (3H, s, CH3); 4.79
(2H, q, Jꢀ7.1 Hz, OCH2); 7.45ꢂ7.62 (6H, m, C6H5, H-
6); 7.98 (1H, d, Jꢀ
8.6 Hz, H-5). 13C-NMR (CDCl3):
dꢀ14.3 (CH3); 17.9 (CH3); 19.4 (CH3); 64.2 (OCH2);
/
236 8C.
time: 30 min. Yield: 52%, m.p.: 267ꢂ
cmꢁ1): nꢀ
2220, 2210 (CN); 1665 (CO); 1600; 1510;
1330; 1230; 805. 1H-NMR (CDCl3): dꢀ
1.52 (3H, t, Jꢀ
7.1 Hz, CH3); 2.53 (3H, s, CH3); 3.56ꢂ3.61 (4H, m,
2CH2N); 4.16ꢂ4.21 (4H, m, 2CH2N); 4.72 (2H, q, Jꢀ
7.1 Hz, OCH2); 6.89, 7.91 (m, 4H, C6H4); 7.40ꢂ7.45
(2H, m, C6H5); 7.60ꢂ7.63 (3H, m, C6H5); 8.06 (1H, s, H-
5). 13C-NMR (CDCl3): dꢀ
14.2 (CH3); 26.1 (CH3); 46.8
/
269 8C. IR (KBr,
/
1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
98.5 (CN); 114.4 (C-3); 116.0; 119.3; 129.0; 129.3; 130.2;
131.9; 133.0; 137.0; 152.1; 155.6; 157.0; 158.1; 162.8;
167.3. MS (EI, m/z, %): 386 [Mꢃ, 100]. Anal.
C22H18N4OS (C, H, N).
/
(2CH2N); 47.1 (2CH2N); 64.4 (OCH2); 94.7 (C-3); 96.1
(C-6); 111.1; 113.3 (CN); 114.2 (CN); 117.2; 128.0;
129.1; 130.4; 130.6; 132.3; 146.8; 153.3; 156.7; 159.1;
165.0; 174.7 (CO). MS (EI, m/z, %): 502 [Mꢃ, 5]. Anal.
C30H26N6O2 (C, H, N).
6.1.1.15. 7-Amino-3-cyano-2-ethoxy-4-phenyl-1,8-
naphthyridine (2n). Reaction time: 52 h. Recrystallised
from ethanol. M.p.: 268ꢂ
cmꢁ1): nꢀ
3480, 3100 (NH); 2220 (CN); 1620; 1570;
1420; 1325; 1010. 1H-NMR (DMSO-d6): dꢀ
1.39 (3H, t,
Jꢀ7.1 Hz, CH3); 4.52 (2H, q, Jꢀ7.1 Hz, OCH2); 6.65
(1H, d, Jꢀ8.1 Hz, H-6); 7.30ꢂ7.59 (8H, m, C6H5, H-5,
NH2); 13C-NMR (DMSO-d6): dꢀ
14.3 9 (CH3); 62.5
(OCH2); 90.4; 110.4; 111.6; 115.6 (CN); 128.4; 128.7;
129.1; 129.5; 133.9; 135.8; 156.3; 157.2; 162.1; 162.6. MS
(EI, m/z, %): 290 [Mꢃ, 100]. Anal. C17H14N4O (C, H,
N).
/
270 8C. Yield: 53%; IR (KBr,
6.1.3.3. 7-Morpholino-3,6-dicyano-2-ethoxy-4-phenyl-
1,8-naphthyridine (5b). Reaction time: 5 h. Yield: 71%;
/
/
m.p.: 255ꢂ
(CN); 1600; 1510; 1420; 1325; 1120; 1020; 810. 1H-NMR
(CDCl3): dꢀ1.52 (3H, t, Jꢀ7.1 Hz, CH3); 3.85ꢂ3.90
(4H, m, CH2NCH2); 3.99ꢂ4.04 (4H, m, CH2OCH2);
4.72 (2H, q, Jꢀ7.1 Hz, OCH2); 7.39ꢂ7.44 (2H, m,
C6H5); 7.60ꢂ
7.63 (3H, m, C6H5); 8.04 (1H, s, H-5). 13C-
NMR (CDCl3): dꢀ14.3 (CH3); 48.1 (NCH2); 64.5
/
256 8C. IR (KBr, cmꢁ1): nꢀ
/2860; 2230, 2210
/
/
/
/
/
/
/
/
/
/
/
/
/
(OCH2); 66.6 (OCH2); 94.9 (C-3); 96.1 (C-6); 111.2;
114.3 (CN); 117.3; 129.2; 129.3; 130.6; 132.4; 146.9;
157.8; 159.4; 165.1. MS (EI, m/z, %): 385 [Mꢃ, 76].
Anal. C22H19N5O2 (C, H, N).
6.1.2. 7-Chloro-3,6-dicyano-2-ethoxy-4-phenyl-1,8-
naphthyridine (4)
A solution of 3 [3] (0.42 g, 1.3 mmol and phosphorus
pentachloride (0.26 g, 1.3 mmol) in phosphorus oxy-
chloride (15 mL) was refluxed for 4 h. The solvent was
removed under reduced pressure. The reaction mixture
was stirred for 12 h with an ice-cooled solution of water
(50 mL) and 10% ammonium hydroxide (15 mL). The
solid was filtered off and recrystallised from ethanol.
6.1.3.4. 3,6-Dicyano-2-ethoxy-7-[4-(4-
nitrophenylpiperazino)]-4-phenyl-1,8-naphthyridine (5c).
Reaction time: 24 h. Yield: 93%.; m.p.: 265ꢂ
/
266 8C. IR
2220 (CN); 1593; 1500; 1445; 1325;
(KBr, cmꢁ1): nꢀ
/
1
1230; 1110; 1015. H-NMR (CDCl3): dꢀ
Jꢀ7.0 Hz, CH3); 3.68 (4H, m, CH2NCH2); 4.21 (4H, m,
CH2NCH2); 4.73 (2H, q, Jꢀ7.0 Hz, OCH2); 6.86, 8.17
(4H, m, C6H4); 7.42ꢂ7.62 (5H, m, C6H5); 8.08 (1H, s, H-
5). 13C-NMR (CDCl3): dꢀ
14.2 (CH3); 46.4
/1.53 (3H, t,
/
Yield: 0.38 g (85%); m.p.: 238ꢂ
nꢀ3060; 2230 (CN); 1580; 1570; 1420, 1355; 1300; 1235;
1150; 1060; 1020; 950. 1H-NMR (CDCl3): dꢀ
1.57 (3H,
t, Jꢀ7.1 Hz, CH3); 4.81 (2H, q, Jꢀ7.1 Hz, OCH2);
7.44ꢂ
7.70 (5H, m, C6H5); 8.30 (1H, s, H-5). 13C-NMR
(CDCl3): dꢀ14.2 (CH3); 65.7 (OCH2); 101.4; 107.7 (C-
/
239 8C. IR (KBr, cmꢁ1):
/
/
/
/
/
/
/
(CH2NCH2); 46.9 (CH2NCH2); 64.5 (OCH2); 112.6
(CN); 117.3; 126.0; 129.2; 129.3; 130.7; 132.5; 139.2;
146.9; 154.3. MS (EI, m/z, %): 505 [Mꢃ, 9]. Anal.
C28H23N7O3 (C, H, N).
/
/
3, C-6); 113.1; 114.2 (CN); 116.0 (CN); 129.1; 129.6;
131.3; 131.4; 144.2; 155.2; 155.9; 158.5; 164.9. MS (EI,
m/z, %): 334 [Mꢃ, 35]. Anal. C18H11N4OCl (C, H, N).
6.1.3.5. 3,6-Dicyano-2-ethoxy-4-phenyl-7-(4-(3-
trifluoromethylphenyl)piperazino]-1,8-naphthyridine
6.1.3. 3,6-Dicyano-2-ethoxy-4-phenyl-1,8-naphthyridines
(5d). Reaction time: 24 h. Yield: 97%; m.p.: 239ꢂ
241 8C. IR (KBr, cmꢁ1): nꢀ
2225 (CN); 1600; 1445;
1400; 1330; 1230; 1110; 800. 1H-NMR (CDCl3): dꢀ
1.53
(3H, t, Jꢀ7.1 Hz, CH3); 3.43ꢂ3.47 (4H, m, CH2NCH2);
4.16ꢂ4.20 (4H, m, CH2NCH2); 4.74 (2H, q, Jꢀ7.1 Hz,
OCH2); 7.10ꢂ7.63 (9H, m, C6H5, C6H4); 8.07 (1H, s, H-
/
(5aꢂo)
/
/
/
6.1.3.1. General procedure. A solution of 4 (0.2 g, 0.60
mmol), the appropriate secondary amine (0.63 mmol)
and triethylamine (0.16 mL, 1.2 mmol) was refluxed in
/
/
/
/
/

272
J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
5). 13C-NMR (CDCl3): dꢀ
/
14.2 (CH3); 47.4 (NCH2);
time: 20 h. Yield: 85%; m.p.: 276ꢂ
cmꢁ1): nꢀ
2220, 2210 (CN); 1595; 1585; 1515; 1325;
1220; 1135; 1020; 800. 1H-NMR (CDCl3): dꢀ
1.53 (3H,
t, Jꢀ7.1 Hz, CH3); 3.39 (4H, br s, CH2NCH2); 4.18
(4H, br s, CH2NCH2); 4.73 (2H, q, Jꢀ7.1 Hz, OCH2);
6.89ꢂ6.99 (3H, m, C6H5); 7.27ꢂ7.63 (7H, m, 2C6H5);
8.05 (1H, s, H-5). 13C-NMR (CDCl3): dꢀ
14.1 (CH3);
/
278 8C. IR (KBr,
48.5 (NCH2); 64.4 (OCH2); 95.0 (C-3); 96.2 (C-6); 111.2;
112.4 (CN); 114.3 (CN); 116.5; 117.3; 119.0; 129.2;
129.8; 130.6; 131.4; 131.9; 132.5; 146.8; 150.9; 156.9;
157.9; 159.4; 165.2. MS (EI, m/z, %): 528 [Mꢃ, 5]. Anal.
C29H23N6OF3 (C, H, N).
/
/
/
/
/
/
/
6.1.3.6. 3,6-Dicyano-2-ethoxy-4-phenyl-7-[4-(2-
pyridylpiperazino)]-1,8-naphthyridine (5e). Reaction
time: 20 h. Yield: 100%; m.p.: 300 8C. IR (KBr,
47.5 (NCH2); 48.9 (NCH2); 64.3 (OCH2); 94.8 (C-3);
95.9 (C-6); 110.0; 114.2 (CN); 116.2; 117.3; 120.3; 129.1;
129.2; 130.5; 132.5; 146.8; 150.6; 156.8; 157.7; 159.2;
165.0. MS (EI, m/z, %): 460 [Mꢃ, 11]. Anal.
C28H24N6O (C, H, N).
cmꢁ1): nꢀ
/
2220 (CN); 1590; 1490; 1430; 1320; 1240;
800. 1H-NMR (CDCl3): dꢀ
1.84 (3H, t, Jꢀ7.1 Hz,
CH3); 4.07ꢂ4.11 (4H, m, CH2NCH2); 4.45ꢂ4.49 (4H, m,
CH2NCH2); 5.04 (2H, q, Jꢀ7.1 Hz, OCH2); 6.98ꢂ7.03
(2H, m, C6H5); 7.72ꢂ7.94 (6H, m, C6H5, C5H4N); 8.36
(1H, s, H-5); 8.53 (1H, d, Jꢀ
6.7 Hz C5H4N). 13C-NMR
(CDCl3): dꢀ14.1 (CH3); 44.6 (CH2NCH2); 47.1
/
/
/
/
/
/
6.1.3.10. 3,6-Dicyano-2-ethoxy-4-phenyl-7-[4-(2-
/
pyrimidinyl)piperazino]-1,8-naphthyridine (5i). Reac-
/
tion time: 22 h. Yield: 91%; m.p.: ꢀ
cmꢁ1): nꢀ
2220 (CN); 1585; 1490; 1440; 1320; 1245;
1135; 975; 800. H-NMR (CDCl3): dꢀ
7.1 Hz, CH3); 4.04ꢂ4.12 (8H, m, CH2NCH2); 4.73 (2H,
q, Jꢀ7.1 Hz, OCH2); 6.58 (1H, t, Jꢀ4.8 Hz, C6H3);
7.40ꢂ7.64 (5H, m, C6H5); 8.06 (1H, s, H-5); 8.37 (2H, d,
Jꢀ 14.3 (CH3);
4.8 Hz, C6H3). 13C-NMR (CDCl3): dꢀ
/
300 8C. IR (KBr,
/
/
1
(CH2NCH2); 64.2 (OCH2); 94.7 (C-3); 95.7 (C-6);
106.8; 110.9; 113.7 (CN); 114.2 (CN); 117.2; 129.1;
130.4; 132.4; 137.5; 146.7; 147.9; 156.7; 157.6; 158.8;
159.2; 165.0. MS (EI, m/z, %): 461 [Mꢃ, 3]. Anal.
C27H23N7O (C, H, N).
/
1.53 (3H, t, Jꢀ
/
/
/
/
/
/
/
43.3 (CH2NCH2); 47.5 (CH2NCH2); 64.4 (OCH2); 94.9
(C-3); 95.9 (C-6); 110.5; 111.1; 114.3 (CN); 117.3; 129.2;
130.6; 132.5; 146.8; 156.8; 157.7; 159.4; 161.4; 165.0. MS
(EI, m/z, %): 462 [Mꢃ, 8]. Anal. C26H22N8O (C, H, N).
6.1.3.7. 3,6-Dicyano-2-ethoxy-7-(4-benzylpiperazino)-4-
phenyl-1,8-naphthyridine (5f). Reaction time: 20 h.
Yield: 93%; m.p.: 229ꢂ
2210 (CN); 1595; 1555; 1500; 1445; 1325; 1250; 1140;
800. 1H-NMR (CDCl3): dꢀ
1.51 (3H, t, Jꢀ7.1 Hz,
CH3); 2.61ꢂ2.65 (4H, m, CH2NCH2); 3.59 (2H, s, CH2);
4.03ꢂ4.05 (4H, m, CH2NCH2); 4.71 (2H, q, Jꢀ7.1 Hz,
OCH2); 7.30ꢂ7.61 (10H, m, 2C6H5); 8.00 (1H, s, H-5).
13C-NMR (CDCl3): dꢀ
14.1 (CH3); 47.6 (NCH2); 52.8
/
230 8C. IR (KBr, cmꢁ1): nꢀ
/
/
/
6.1.3.11. 3,6-Dicyano-2-ethoxy-4-phenyl-7-(4-
/
piperidylpiperidino)-1,8-naphthyridine (5j). Reaction
/
/
time: 16 h. Yield: 95%; m.p.: 215ꢂ
/
217 8C. IR (KBr,
/
cmꢁ1): nꢀ
/
2930; 2220, 2210 (CN); 1595; 1515; 1325;
1
/
1015; 800. H-NMR (CDCl3): dꢀ
piper); 2.00ꢂ2.17 (3H, m, piper); 2.53ꢂ
piper); 3.13 (2H, dt, Jꢀ11.5 Hz, piper); 4.70 (2H, q, Jꢀ
7.1 Hz, OCH2); 4.80ꢂ4.85 (2H, m, piper); 7.38ꢂ7.42
(2H, m, C6H5); 7.57ꢂ7.60 (3H, m, C6H5); 7.98 (1H, s, H-
5). 13C-NMR (CDCl3): dꢀ
14.3 (CH3); 24.6, 26.1, 28.0,
/
1.44ꢂ
/
1.77 (11H, m,
(NCH2); 62.7 (CH2C6H5); 64.2 (OCH2); 94.7 (C-3); 95.6
(C-6); 110.9; 114.4 (CN); 117.4; 127.3; 128.3; 129.2;
130.5; 132.6; 137.5; 146.9; 156.9; 157.7; 159.1; 165.1. MS
(EI, m/z, %): 474 [Mꢃ, 8]. Anal. C29H26N6O (C, H, N).
/
/2.62 (4H, m,
/
/
/
/
/
/
6.1.3.8. 3,6-Dicyano-2-ethoxy-7-[(4-N-
isopropylacetamido)piperazino]-4-phenyl-1,8-
napthyridine (5g). Reaction time: 30 min. Yield: 93%;
40.8, 47.7, 50.1, 62.2 (piper); 64.3 (OCH2); 94.9 (C-3);
95.4 (C-6); 110.8; 114.5 (CN); 117.4; 129.2; 130.5; 132.6;
146.8; 157.0; 157.6; 159.1; 165.0. MS (EI, m/z, %): 466
[Mꢃ, 3]. Anal. C28H30N6O (C, H, N).
m.p.: 265ꢂ
2220, 2210 (CN); 1645 (CO); 1595; 1325; 1010. 1H-
NMR (CDCl3): dꢀ1.19 (6H, d, Jꢀ6.5 Hz CH3); 1.52
(3H, t, Jꢀ7.0 Hz, CH3); 2.70ꢂ2.71 (4H, m, CH2NCH2);
3.06 (2H, s, CH2CO); 4.01ꢂ4.17 (5H, m, CH2NH2, CH);
4.71 (2H, q, Jꢀ7.0 Hz, OCH2); 6.84 (1H, br s, NH);
7.39ꢂ7.42 (2H, m, C6H5); 7.60ꢂ7.62 (3H, m, C6H5); 8.03
(1H, s, H-5). 13C-NMR (CDCl3): dꢀ
14.2 (CH3); 22.7 (2
/
266 8C. IR (KBr, cmꢁ1): nꢀ
/3280 (NH);
/
/
6.1.3.12. 3,6-Dicyano-2-ethoxy-4-phenyl-7-(4-
/
/
phenylpiperidino)-1,8-naphthyridine (5k). Reaction
/
time: 16 h. Yield: 95%; m.p.: 273ꢂ
cmꢁ1): nꢀ
2910; 2220 (CN); 1595; 1470; 1450; 1325;
1240; 1005; 800. 1H-NMR (CDCl3): dꢀ
1.45 (3H, t, Jꢀ
7.1 Hz, CH3); 1.79ꢂ2.03 (4H, m, piper); 2.79ꢂ2.95 (1H,
m, piper); 3.19 (2H, dd, Jꢀ11.9 Hz, piper); 4.65 (2H, q,
Jꢀ7.1 Hz, OCH2); 4.88 (2H, dd, Jꢀ13.5 Hz, piper);
7.16ꢂ
7.56 (10H, m, 2C6H5); 7.95 (1H, s, H-5). 13C-
NMR (CDCl3): dꢀ14.3 (CH3); 33.3, 42.6, 48.8 (piper);
/
275 8C. IR (KBr,
/
/
/
/
/
/
/
/
/
CH3); 40.7 (NCH2); 47.7 (NCH2); 53.1 (CH2); 61.4
(CH); 64.4 (OCH2); 95.1 (CN); 97.2 (CN); 111.2; 114.3
(CN); 117.3; 129.2; 129.3; 130.6; 132.5; 146.8; 157.8;
159.5; 165.1; 168.4 (CO). MS (EI, m/z, %): 483 [Mꢃ, 6].
Anal. C27H29N7O2 (C, H, N).
/
/
/
/
/
64.3 (OCH2); 95.0 (C-3); 95.5 (C-6); 110.8; 114.5 (CN);
117.5; 126.6; 126.8; 128.6; 129.2; 130.5; 132.6; 144.9;
146.9; 157.0; 157.7; 159.4; 165.1. MS (EI, m/z, %): 459
[Mꢃ, 39]. Anal. C29H25N5O (C, H, N).
6.1.3.9. 3,6-Dicyano-2-ethoxy-4-phenyl-7-(4-
phenylpiperazino)-1,8-naphthyridine (5h). Reaction

J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ
/
275
273
6.1.3.13. 7-(4-Benzylpiperidino)-3,6-dicyano-2-ethoxy-
4-phenyl-1,8-naphthyridine (5l). Reaction time: 16 h.
NMR (CDCl3): dꢀ
(4H, t, Jꢀ
NCH2); 4.72 (2H, q, Jꢀ
m, C6H5); 7.55ꢂ7.79 (3H, m, C6H5); 8.02 (1H, m, H-5).
13C-NMR (DMSO-d6): dꢀ
14.3 (CH3); 46.0 (NCH2);
49.0 (NCH2); 64.4 (OCH2); 94.9 (C-3); 95.9 (C-6); 110.9,
114.4 (CN); 117.4; 129.2, 130.6, 132.5 (C6H5); 146.9 (C-
5); 157.7; 159.4; 165.1. MS (EI, m/z, %): 384 [Mꢃ, 2].
Anal. C22H20N6O (C, H, N).
/
1.53 (3H, t, Jꢀ
/
7.0 Hz, CH3); 3.07
/
5.0 Hz, NCH2); 3.99 (4H, t, Jꢀ
/
5.0 Hz,
Yield: 91%; m.p.: 204ꢂ
/
205 8C. IR (KBr, cmꢁ1): nꢀ
2910; 2220, 2210 (CN); 1600; 1450; 1325; 1130; 1015;
/
/
7.0 Hz, OCH2); 7.39ꢂ7.44 (2H,
/
/
1
795. H-NMR (CDCl3): dꢀ
1.51 (3H, t, Jꢀ7.1 Hz, CH3); 1.84ꢂ
2.61 (2H, d, Jꢀ6.6 Hz, CH2); 3.09 (2H, t, Jꢀ
piper); 4.66ꢂ4.79 (2H, m, piper); 4.70 (2H, q, Jꢀ
OCH2); 7.15ꢂ7.61 (10H, m, 2C6H5); 7.98 (1H, s, H-5).
13C-NMR (CDCl3): dꢀ
14.3 (CH3); 32.1 (CH2); 38.2,
/
1.41ꢂ
/
1.63 (2H, m, piper);
1.88 (3H, m, piper);
12.6 Hz,
7.1 Hz,
/
/
/
/
/
/
/
/
/
42.9, 48.4 (piper); 64.2 (OCH2); 94.9 (C-3); 95.3 (C-6);
110.7; 114.5 (CN); 117.5; 126.1; 128.3; 129.0; 129.1;
130.5; 132.6; 139.8; 146.8; 157.0; 157.6; 159.2; 165.2. MS
(EI, m/z, %): 473 [Mꢃ, 39]. Anal. C30H27N5O (C, H,
N).
6.1.4. 6-Cyano-2-dimethylamino-4-piperazino-7-
thiomorpholinopyrido[2,3-d]pyrimidine (6)
Obtained by a previously reported procedure [11].
Yield: 60%; m.p.: 300 8C. IR (KBr, cmꢁ1). nꢀ
/
2200
(CN); 1600; 1575; 1550; 1500; 1440; 1425. 1H-NMR
(CDCl3). dꢀ2.77 (4H, m, CH2S); 3.18 (s, 6H, NMe2);
/
3.23 (m, 4H, NCH2); 3.85 (m, 8H, NCH2); 8.47 (s, 1H,
H-5); 9.50 (broad singlet exchangeable with D2O, 1H,
6.1.3.14. 3,6-Dicyano-2-ethoxy-7-(4-
ethoxycarbonylpiperazino)-4-phenyl-1,8-naphthyridine
NH). 13C-NMR (DMSO-d6): dꢀ
26.5 (CH2S); 37.1
/
(5m). Reaction time: 24 h. Yield: 65%; m.p.: 273ꢂ
275 8C. IR (KBr, cmꢁ1): nꢀ
2990; 2220, 2210 (CN);
1700 (CO); 1595; 1430; 1245; 1010; 800. 1H-NMR
(CDCl3): dꢀ1.29 (3H, t, Jꢀ7.1 Hz, CH3); 1.52 (3H,
t, Jꢀ7.1 Hz, CH3); 3.67ꢂ3.72 (4H, m, NCH2); 3.96ꢂ
4.00 (4H, m, NCH2); 4.20 (2H, q, Jꢀ7.1 Hz, OCH2);
4.72 (2H, q, Jꢀ7.1 Hz, OCH2); 7.39ꢂ7.44 (2H, m,
C6H5); 7.59ꢂ
7.63 (3H, m, C6H5); 8.05 (1H, s, H-5). 13C-
NMR (CDCl3): dꢀ14.3 (CH3); 14.6 (CH3); 43.3
/
(NMe2); 42.3; 45.6; 49.9 (NCH2); 87.4 (C-6); 97.9 (C-
4a); 118.1 (CN); 144.9 (C-5); 160.1; 163.1. MS (EI, m/z,
%): 384 [Mꢃ, 19]. Anal. C18H24N8S (C, H, N).
/
/
/
/
/
/
/
6.1.5. Synthesis of Pyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-
/
/
triazines (12aꢂp)
/
/
/
6.1.5.1. General procedure. To an ice-cooled solution of
the corresponding b-enaminonitrile (10) [14] (6.73
(NCH2); 47.6 (NCH2); 61.8 (OCH2); 64.5 (OCH2);
95.1 (C-3); 96.2 (C-6); 112.2; 114.3 (CN); 117.2; 129.1;
129.2; 129.6; 130.6; 132.4; 146.8; 155.4; 156.8; 157.8;
159.6; 165.1. MS (EI, m/z, %): 456 [Mꢃ, 21]. Anal.
C25H24N6O3 (C, H, N).
mmol) in 1:1 (v/v) HClꢂ
/
AcOH or HBrꢂAcOH (50
/
mL) a solution of sodium nitrite (10.1 mmol) in water
(10 mL) was added dropwise. The solution was stirred at
room temperature for 3 h, and the mixture was poured
into water (200 mL). The resulting solid was filtered off
and purified by flash chromatography on silica gel to
yield 11. A solution of the corresponding halogenated
derivative 11 (0.60 mmol) and the appropriate second-
6.1.3.15. 3,6-Dicyano-2-ethoxy-7-(4-(2-oxo-2-
morpholinoethyl)piperazino)-4-phenyl-1,8-naphthyridine
(5n). Reaction time: 3 h. Yield: 93%; m.p.: 211ꢂ
IR (KBr, cmꢁ1): nꢀ
2220 (CN); 1635 (CO); 1595; 1500;
1440; 1325; 1110; 1000. 1H-NMR (CDCl3): dꢀ
1.51
(3H, t, Jꢀ7.1, Hz, CH3); 2.71ꢂ2.75 (4H, m,
CH2NCH2); 3.27 (2H, s, CH2CO); 3.63ꢂ3.67 (8H, m,
CH2NCH2, CH2OCH2); 4.01ꢂ4.03 (4H, m, CH2NCH2);
4.69 (2H, q, Jꢀ7.1 Hz, OCH2); 7.38ꢂ7.61 (5H, m,
C6H5); 8.01 (1H, s, H-5). 13C-NMR (CDCl3): dꢀ
14.2
/
213 8C.
/
ary amine (0.72 mmol) in 3:1(v/v) THFꢂEtOH (15 mL)
/
/
was refluxed until the starting material disappeared
(TLC). After cooling, the solid was filtered off and
recrystallised or purified by flash chromatography.
Compounds 12a, 12b, 12d, 12l and 12o have already
been described [14].
/
/
/
/
/
/
/
(CH3); 42.1 (CH2CO); 46.2 (CH2NCH2); 47.6
(CH2NCH2); 52.8; 60.4; 64.4 (OCH2); 66.8 (CH2OCH2);
66.9 (CH2OCH2); 94.9 (C-3); 95.9 (C-6); 111.0; 114.3
(CN); 117.3; 129.1; 129.2; 130.5; 132.4; 146.8; 156.8;
157.7; 159.2; 165.0; 167.5 (CO). MS (EI, m/z, %): 511
[Mꢃ, 3]. Anal. C28H29N7O3 (C, H, N).
6.1.5.2. 8-Cyano-4-(4-methylpiperazino)-7-
morpholinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12c). Reaction time: 3 h. Recrystallised from EtOHꢂ
acetone. M.p.: 294ꢂ296 8C. Yield: 74%; IR (KBr,
cmꢁ1): nꢀ2220 (CN); 1590; 1540; 1275. 1H-NMR
(DMSO-d6): dꢀ2.77 (3H, s, CH3); 3.79 (16H, m,
NCH2, CH2O); 9.10 (1H, s, H-9). 13C-NMR (DMSO-
d6): dꢀ42.3 (CH3); 48.2 (NCH2, CH2); 51.8 (NCH2);
/
/
/
/
6.1.3.16. 3,6-Dicyano-2-ethoxy-4-phenyl-7-piperazino-
1,8-naphthyridine (5o). Reaction time: 30 min. Yield:
/
65.8 (CH2O); 93.8 (C-8); 111.7 (C-4a); 115.9 (CN); 117.4
(C-9a); 140.8 (C-9); 149.1; 152.5; 160.0; 163.8. MS
(FAB, m/z, %): 397 [(MH)ꢃ, 100]. Anal. C18H20N8OS
(C, H, N).
70%; m.p.: 182ꢂ
/
184 8C. IR (KBr, cmꢁ1): nꢀ
3434
(NH); 2224 (CN); 1586; 1571; 1542; 1513; 1445; 1423;
/
1404; 1384; 1329; 1255; 1231; 1133; 1113; 1018. 1H-

274
J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ275
/
6.1.5.3. 4-Chloro-8-cyano-7-
159.8; 163.9. MS (FAB, m/z, %): 395 [(MH)ꢃ, 100].
Anal. C19H22N8S (C, H, N).
thiomorpholinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-
triazine (12e). Reaction time: 0.5 h. Purified by flash
chromatography using CH2Cl2 as eluent. Yield: 94%.
6.1.5.8. 4-Benzylamino-8-cyano-7-
piperidinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
1H-NMR (CDCl3): dꢀ
4.31 (4H, t, Jꢀ
NMR (CDCl3): dꢀ
/
2.88 (4H, t, Jꢀ
5.1 Hz, NCH2); 8.93 (1H, s, H-9). 13C-
27.4 (SCH2); 51.1 (NCH2); 94.1 (C-
/
5.1 Hz, SCH2);
/
(12j). Reaction time: 13 h. Recrystallised from EtOHꢂ
acetone. Yield: 50%. IR (KBr, cmꢁ1): nꢀ
3635 (NH);
2220 (CN); 1595; 1540; 1510; 1495; 1430; 1300; 1250.
1H-NMR (CDCl3): dꢀ
1.77 (6H, m, CH2); 3.87 (4H, m,
NCH2); 5.02 (2H, d, Jꢀ5.7 Hz, NHCH2); 5.41 (1H, t,
Jꢀ5.7 Hz, NH); 7.32ꢂ7.48 (5H, m, C6H5); 8.78 (1H, s,
H-9). 13C-NMR (CDCl3): dꢀ
24.3, 25.9 (CH2); 45.6,
/
/
/
8); 115.3 (C-4a); 116.9 (CN); 129.6 (C-9a); 141.6 (C-9);
150.9; 152.4; 159.8; 165.6. MS (FAB, m/z, %): 317
[(MH)ꢃ, 100]. Anal. C13H9N6S2Cl (C, H, N).
/
/
/
/
6.1.5.4. 8-Cyano-4-piperazino-7-
thiomorpholinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-
triazine (12f). Reaction time: 1 h. Recrystallised from
/
49.5 (NCH2); 93.3 (C-8); 112.0 (C-4a); 116.5 (CN); 117.7
(C-9a); 128.1, 128.2, 128.9, 137.3 (C6H5); 140.5 (C-9);
151.9; 159.9; 164.9. MS (EI, m/z, %): 401 [Mꢃ, 4]. Anal.
C21H19N7S (C, H, N).
EtOH. M.p.: 192ꢂ
/
194 8C. Yield: 84%; 1H-NMR
(DMSO-d6): dꢀ2.82 (4H, m, SCH2); 4.09ꢂ4.18 (8H,
/
/
m, NCH2); 9.03 (1H, b.s., H-9); 9.56 (1H, b.s. exchange-
able with D2O, NH). MS (FAB, m/z, %): 399 [(MH)ꢃ,
100]. Anal. C17H18N8S2 (C, H, N).
6.1.5.9. 8-Cyano-7-(4-methylpiperidino)-4-
piperazinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12k). Reaction time: 1 h. Recrystallised from EtOHꢂ
acetone. M.p.: 213ꢂ215 8C. Yield: 97%; IR (KBr,
cmꢁ1): nꢀ
3400 (NH); 2215 (CN); 1597; 1551; 1533;
1495; 1445; 1405; 1364; 1314; 1276; 1258; 1245. 1H-
NMR (DMSO-d6): dꢀ0.94 (3H, d, Jꢀ6.0 Hz, CH3);
1.27 (2H, m, CH2); 1.77 (3H, m, CH2, CH); 2.90 (4H, m,
NCH2); 3.13 (2H, m, NCH2); 3.89 (4H, m, NCH2); 4.42
(2H, m, NCH2); 8.85 (1H, s, H-9). 13C-NMR (DMSO-
/
6.1.5.5. 8-Cyano-4,7-
/
dipiperidinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
/
(12g). Reaction time: 5 h. Recrystallised from EtOHꢂ
acetone. M.p.: 228ꢂ230 8C. Yield: 68%; IR (KBr,
cmꢁ1): nꢀ
2220 (CN); 1600; 1550ꢂ1535; 1500; 1450;
1360; 1310; 1270; 1245. 1H-NMR (CDCl3): dꢀ
1.79
(12H, m, CH2); 3.86 (4H, m, NCH2); 4.03 (4H, m,
NCH2); 8.79 (1H, s, H-9). 13C-NMR (CDCl3): dꢀ
24.3,
/
/
/
/
/
/
/
/
d6): dꢀ21.5 (CH3); 30.1 (CH); 33.6, 42.8 (CH2); 46.2,
/
24.4, 25.8, 26.0 (CH2); 47.3, 49.5 (NCH2); 93.2 (C-8);
110.9 (C-4a); 116.1 (CN); 117.7 (C-9a); 140.2 (C-9);
148.9; 152.5; 160.0; 164.4. MS (EI, m/z, %): 379 [Mꢃ,
19]. Anal. C19H21N7S (C, H, N).
48.3 (NCH2); 93.1 (C-8); 110.4 (C-4a); 115.5 (CN); 117.5
(C-9a); 140.3 (C-9); 148.5; 152.3; 159.7; 163.8. MS (EI,
m/z, %): 394 [Mꢃ, 6]. Anal. C19H22N8S (C, H, N).
6.1.5.10. 7-(4-Benzylpiperazino)-8-cyano-4-
piperazinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12m). Reaction time: 0.5 h. Recrystallised from EtOH.
6.1.5.6. 8-Cyano-4-piperazino-7-
piperidinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12h). Reaction time: 1 h. Recrystallised from EtOH.
M.p.: 215ꢂ
nꢀ2220 (CN); 1600; 1550; 1530; 1490; 1440; 1360ꢂ
1340; 1310; 1270; 1250. 1H-NMR (DMSO-d6): dꢀ
/
217 8C (dec.). Yield: 66%; IR (KBr, cmꢁ1):
1
M.p.: 281ꢂ
dꢀ
m, NCH2); 8.99 (1H, s, H-9); 9.46 (1H, b.s., NH). 13C-
NMR (DMSO-d6): dꢀ23.7, 25.4 (CH2); 42.2, 42.4, 49.0
/
283 8C. Yield: 80%; H-NMR (DMSO-d6):
/
/
/1.68 (6H, m, CH2); 3.81 (4H, m, NCH2); 4.17 (4H,
/
2.56 (4H, b.s., NCH2); 3.23 (4H, m, NCH2); 3.56 (2H,
s, CH2); 3.82 (4H, m, NCH2); 4.11 (4H, m, NCH2); 7.33
(5H, m, C6H5); 9.02 (1H, s, H-9). Anal. C24H25N9S (C,
H, N).
/
(NCH2); 93.2 (C-8); 111.3 (C-4a); 115.1 (CN); 117.5 (C-
9a); 140.6 (C-9); 149.0; 152.4; 159.8; 163.9. MS (FAB,
m/z, %): 381 [(MH)ꢃ, 100]. Anal. C18H20N8S (C, H, N).
6.1.5.11. 7-Benzylamino-8-cyano-4-
dimethylaminopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-
triazine (12n). Reaction time: 2 h. Purified by flash
6.1.5.7. 8-Cyano-4-(4-methylpiperazino)-7-
piperidinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12i). Reaction time: 3 h. Recrystallised from EtOHꢂ
acetone. M.p.: 270 8C (dec.). Yield: 70%; IR (KBr,
cmꢁ1): nꢀ
2240 (CN); 1610; 1560; 1540; 1510; 1460;
1420; 1410; 1370; 1320; 1280; 1260. H-NMR (DMSO-
d6): dꢀ1.68 (6H, m, CH2); 2.79 (3H, s, CH3); 3.79 (4H,
m, NCH2); 4.14ꢂ4.29 (8H, m, NCH2); 8.91 (1H, s, H-9).
13C-NMR (DMSO-d6): dꢀ
23.7, 25.4 (CH2); 42.2
/
chromatography using as eluent CH2Cl2ꢂ
(v/v). M.p.: 198ꢂ
200 8C. Yield: 60%; IR (KBr, cmꢁ1):
nꢀ3351 (NH); 2217 (CN); 1597; 1560; 1518; 1453; 1428;
1382; 1345; 1323; 1297; 1239; 1114; 1084. 1H-NMR
(DMSO-d6): dꢀ3.34 (6H, s, NCH3); 4.63 (2H, d, Jꢀ
5.4 Hz, CH2); 7.21ꢂ7.34 (5H, m, C6H5); 8.57 (1H, m,
NH); 8.74 (1H, s, H-9). 13C-NMR (DMSO-d6): dꢀ
38.3
/
AcOEt 20:1
/
/
/
1
/
/
/
/
/
/
/
(CH3); 42.6, 49.0, 51.6 (NCH2); 93.2 (C-8); 111.3 (C-
4a); 115.0 (CN); 117.5 (C-9a); 140.6 (C-9); 149.1; 152.3;
(NCH3); 44.5 (CH2); 91.5 (C-8); 109.3 (C-4a); 114.2
(CN); 116.0 (C-9a); 126.8, 127.4, 128.3, 138.9 (C6H5);

J.M. Quintela et al. / European Journal of Medicinal Chemistry 38 (2003) 265ꢂ
/
275
275
138.2 (C-9); 148.1; 152.7; 158.1; 165.1. Anal. C18H15N7S
(C, H, N).
as negative controls. In order to rule out possible effects
of the solvent in samples dissolved in DMSO, duplicate
wells with PBS or filtered sea water containing the
highest concentration of DMSO used (up to 2.5%) were
also included.
6.1.5.12. 8-Cyano-7-diethylamino-4-
morpholinopyrido[3?,2?:4,5]thieno[3,2-d]-1,2,3-triazine
(12p). Reaction time: 3 h. Recrystallised from EtOHꢂ
acetone. M.p.: 229ꢂ231 8C. Yield: 83%; IR (KBr,
cmꢁ1): nꢀ
2220 (CN); 1600; 1560; 1510; 1450; 1430;
1385; 1360; 1335; 1320; 1270; 1250. H-NMR (CDCl3):
dꢀ1.36 (6H, t, Jꢀ7.1 Hz, CH3); 3.82ꢂ3.92 (8H, m,
NCH2); 4.04 (4H, m, OCH2); 8.78 (1H, s, H-9). 13C-
NMR (CDCl3): dꢀ13.4 (CH3); 44.9, 46.0 (NCH2); 66.5
/
Ciliate motility after incubation was checked using an
inverted microscope with phase-contrast illumination.
Prior to scanning, each culture plate was gently rocked
to ensure uniform distribution of ciliates throughout the
medium. The results are expressed as LD: the minimum
lethal concentration required to kill 99% of the ciliates.
/
/
1
/
/
/
/
(OCH2); 90.1 (C-8); 110.9 (C-4a); 114.5, 118.5 (CN, C-
9a); 141.0 (C-9); 149.5; 152.8; 157.2; 164.8. MS (EI, m/z,
%): 369 [Mꢃ, 88]. Anal. C17H19N7OS (C, H, N).
Acknowledgements
This work was supported by grants from CICYT
(PM98-0227, FEDER-CICYT IFD97-2157 and IFD97-
0032) and from Xunta de Galicia (XUGA-PGIDT99-
BIO20901).
6.2. Pharmacology
6.2.1. Isolation and culture of P. dicentrarchi
Ciliates were harvested by collecting ascitic fluid from
the body cavity of naturally infected turbot and were
maintained under the culture conditions described by
Bernard and Fenchel [15] with autoclaved V. anguil-
larum as food.
References
[1] P.J. Cheung, R.F. Nigrelli, D. Ruggieri, J. Fish. Dis. 3 (1980)
295ꢂ/303.
[2] J.F. Morado, E.B. Small, Rev. Fish. Sci. 3 (1995) 275ꢂ
[3] R.J. Cawthorn, D.H. Lynn, B. Despres, R. MacMillan, R.
Maloney, M. Loughlin, R. Bayer, Dis. Aquat. Org. 24 (1996)
/
354.
6.2.2. Determination of lethal activity
Stock solutions of the synthesised compounds were
prepared in distilled water or dimethylsulphoxide
(DMSO; Sigma Chemical Co., St. Louis, MO, USA).
The resulting stocks were diluted in physiological
phosphate-buffered saline (PBS; pH 7.2) or filtered
(0.2 mm) sea water (salinity 28%) to give the final
concentrations used in the screening (see below). Tests
performed in filtered sea water allow automatic exclu-
sion of the possibility of partial or total inactivation of
the test substance under marine conditions.
143ꢂ
[4] B.L. Munday, P.J. O’Donoghue, M. Watts, K. Rough, T.
Hawkesford, Dis. Aquat. Org. 30 (1997) 17ꢂ25.
[5] A. Dragesco, J. Dragesco, F. Coste, C. Gasc, B. Romestand, J.
Raymond, G. Bouix, Eur. J. Protistol. 31 (1995) 327ꢂ340.
[6] R. Iglesias, A. Parama´, M.F. Alvarez, J. Leiro, J. Ferna´ndez,
M.L. Sanmart´ın, Dis. Aquat. Org. 46 (2001) 47ꢂ55.
[7] R. Iglesias, A. Parama´, M.F. .Alvarez, J. Leiro, M.L. Sanmart´ın,
Dis. Aquat. Org. 49 (2002) 191ꢂ197.
/148.
/
/
/
´
/
[8] G.P. Ellis, in: E.C. Taylor (Ed.), The Chemistry of Heterocyclic
Compounds, vol. 47 (2) (and references therein), Wiley-Inter-
Ciliates in the late exponential phase or early plateau
phase of culture were concentrated by centrifugation at
science, Chichester, UK, 1992, pp. 670ꢂ674.
/
[9] J.M. Quintela, J.L. Soto, Ann. Quim. 80C (1984) 268 (Chem.
650ꢄg for 5 min and then resuspended in PBS or
/
Abstr. 103 (1985) 37345).
[10] J.M. Quintela, J. Vilar, C. Peinador, C. Veiga, V. Ojea, Hetero-
filtered sea water. After counting in a haemocytometer,
10 mL of ciliate suspension containing 104 ciliates was
added to each well of a 96-well microtitre polystyrene
plate containing 90 mL per well of the candidate
antiprotozoal at the required dose (100, 50, 25, 12.5,
6.2, 3.1, 1.5 and 0.7 mg Lꢁ1) in PBS or filtered sea
water. Plates were then incubated at 18 8C for 24 h.
Each determination was performed in duplicate.
Wells containing ciliates in assay solution (PBS or
filtered sea water) without chemicals were also assayed
cycles 41 (1995) 1001ꢂ
[11] J.M. Quintela, C. Peinador, L. Botana, M. Este´vez, R. Riguera,
Bioorg. Med. Chem. 5 (1997) 1533ꢂ1543.
[12] J.M. Quintela, C. Peinador, C. Veiga, L. Gonza´lez, L. Botana, A.
Alfonso, R. Riguera, Bioorg. Med. Chem. 6 (1998) 1911ꢂ1925.
[13] J.M. Quintela, C. Peinador, C. Veiga, L. Botana, A. Alfonso, R.
Riguera, Eur. J. Med. Chem. 33 (1998) 887ꢂ897.
/
1011.
/
/
/
[14] J.M. Quintela, C. Peinador, L. Gonza´lez, R. Riguera, I. Rioja,
M.C. Terencio, A. Ubeda, M.J. Alcaraz, J. Med. Chem. 42 (1999)
4720ꢂ/4724.
[15] C. Bernard, T. Fenchel, Eur. J. Protistol. 32 (1996) 293ꢂ
/
297.